JP7235785B2 - Vaccine against hepatitis B virus - Google Patents

Description

The present invention relates to immunogenic HBV peptide compositions and treatment of HBV using such compositions.

Hepatitis B virus (HBV) infection is a major cause of liver-related morbidity and mortality in Europe and worldwide. An estimated 650,000 individuals die each year from liver failure or hepatocellular carcinoma. Chronic hepatitis B (CHB) is increasing rapidly in Europe due to the immigration of HBV carriers from endemic areas, despite vaccination programs in many countries resulting in a decline in new HBV infections. is a problem.

From a conceptual point of view, chronic HBV infection can be divided into three stages (or types of immune response): immune-tolerant, immune-active and immune-inactive chronic carriers. These distinct phases of chronic infection correspond to characteristic serological patterns and correlate with the patient's immune response to HBV. Generally, patients with persistent immunoreactive chronic HBV infection receive HBV therapy.

Limited treatment options are available for chronic hepatitis B (CHB). Suppression of viral replication with antiviral agents such as interferon-alpha and nucleoside/nucleotide analogues (NUCs) is the only way to reduce the morbidity and mortality of chronic HBV infection with the ultimate goal of improving survival. is. Nonetheless, the disappearance of serum HBsAg and the development of anti-HBs antibodies (seroconversion) are evidence of a successful immunological response to HBV infection and are the outcomes closest to clinical cure. be. Only interferon-alpha was able to induce significant HBsAg loss, however, in a relatively low percentage of patients (<10%). Interferons are expensive and poorly tolerated, and some HBV genotypes remain poorly responsive to treatment.

Therefore, NUC remains the main therapeutic strategy, with 5 NUCs approved to treat CHB in Europe. The most potent and preferred drugs, tenofovir and entecavir, have very favorable side effect profiles and are able to induce HBV DNA suppression in almost all patients. However, for the majority of patients, lifelong therapy is required under most national and international guidelines. Even after several years of NUC therapy, only a minority of HBeAg-positive patients and no HBeAg-negative patients are able to clear HBsAg. The long-term safety of NUC therapy is currently unknown. Therefore, there is an urgent need for initiatives that allow timely discontinuation of NUC therapy.

Therapeutic vaccination is a promising intervention for hepatitis B as a way to induce immune control against the disease. T cell responses have been shown to be important for clearance of acute HBV infection. However, HBsAg-based therapeutic HBV vaccines have failed to show benefit from immune tolerance induced from high levels of circulating HBsAg, even under effective antiviral therapy.

We identified HBV proteomic regions with a high degree of conservation among different HBV genotypes and unexpectedly good immunogenic properties compared to other similarly conserved regions of HBV proteins. identified. In particular, we have unexpectedly discovered, using in vitro assays, that specific domains of HBV polymerase and peptide sequences within the HBV core protein were isolated from chronically infected HBV patients with different infected HBV genotypes. and/or PBMC from chronically infected HBV patients of different ethnicities. In particular, the inventors have surprisingly identified an immunodominant region within the terminal domain of HBV polymerase.

Thus, in certain embodiments, the present invention provides for one of the sequences set forth in SEQ ID NOs: 1-4 or at least 80% A pharmaceutical composition comprising at least two peptides of 15 to 60 amino acids in length selected from peptides comprising a sequence of at least 15 contiguous amino acids of a sequence having the identity of comprising at least one CD8+ T cell epitope and/or at least one CD4+ T cell epitope, each peptide eliciting a response in peripheral blood mononuclear cells (PBMC) from at least one individual with chronic HBV infection in an in vitro assay; A pharmaceutical composition is provided that induces.

The composition comprises at least one peptide comprising at least 15 amino acids of one of the sequences set forth in SEQ ID NOS: 1-3 and at least 15 amino acids of the sequence set forth in SEQ ID NO:4. may comprise at least one peptide comprising

At least one of the peptides is a sequence set forth in one of SEQ ID NOS: 24-33, or at least 80% of one of the sequences set forth in SEQ ID NOS: 24-33. It may contain sequences of identity. One or more of the peptides contain one or more amino acid(s) at the N-terminus and/or C-terminus to increase the net positive charge of the peptide and/or to reduce the hydrophobicity. OK. Accordingly, a composition may comprise a peptide comprising a sequence set forth in one of SEQ ID NOs:34-38.

The composition may further comprise at least one peptide derived from HBV surface protein. Peptides derived from HBV surface proteins are 15-60 amino acids in length and are sequences of at least 15 contiguous amino acids of the sequence shown in SEQ ID NO:55, or of the sequence shown in SEQ ID NO:55. may comprise a sequence having at least 80% identity over at least 15 contiguous amino acids, wherein said peptide comprises at least one CD8+ T cell epitope and/or at least one CD4+ T cell epitope and elicits a response in peripheral blood mononuclear cells (PBMC) from at least one individual with chronic HBV infection in an in vitro assay.

The composition is capable of inducing an immune response in PBMC from at least two individuals of different ethnicity and PBMC from two individuals with different HBV genotypes being infected.

The composition comprises (a) an individual infected with HBV genotype A, an individual infected with HBV genotype B, an individual infected with HBV genotype C, and an individual infected with HBV genotype D PBMCs from 2, 3 or all of the individuals and/or HBV-infected Asian or Indian individuals, HBV-infected Caucasian individuals, and HBV-infected individuals It is possible to induce an immune response in PBMC from 2, 3 or all African or Arab individuals.

Peptides in the compositions of the invention may be linked to a fluorocarbon vector. The composition may further comprise an HBc, HBe or HBs antigen and/or an adjuvant.

The present invention provides compositions of the invention for use in the treatment or prevention of HBV infection, particularly for treating HBeAg-negative or HBeAg-positive patients. The compositions of the invention contain (i) interferon-alpha and/or nucleoside/nucleotide analogues (NUC), and/or (ii) anti-PD1, anti-CTLA4, anti-PD1L, anti-LAG3 blocking antibodies. , anti-TIM3 blocking antibodies and/or cyclophosphamide. Treatment with the composition may result in HBsAg loss or HBsAg seroconversion.

The invention also provides compositions of the invention for use in treating or preventing end-stage liver disease or hepatocellular carcinoma, or for use in treating or preventing hepatitis D virus (HDV) infection.

A method of treating or preventing HBV infection comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to the present invention, and a pharmaceutical for treating or preventing HBV. The use of the composition according to the invention in manufacturing is also provided.

Furthermore, the present invention has at least 80% identity to the sequence set forth in any one of SEQ ID NOs: 1-4 or to one of the sequences set forth in SEQ ID NOs: 1-4 A peptide 15 to 60 amino acids in length comprising at least 15 contiguous amino acids of a sequence and comprising at least one CD8+ T cell epitope and/or at least one CD4+ T cell epitope, wherein in an in vitro assay, Peptides are provided that are capable of inducing a response in peripheral blood mononuclear cells (PBMC) from at least one individual with chronic HBV infection. A peptide may comprise at least 15 contiguous amino acids of the sequence shown in SEQ ID NO:5, 6, 14 or 15.

The present invention provides sequences having at least 80% identity to one of the sequences set forth in SEQ ID NOs:24-38 or to one of the sequences set forth in SEQ ID NOs:24-38. Also provided are peptides comprising:

Peptides of the invention may be covalently linked to a fluorocarbon vector.

FIG. 10 is a graph showing a comparison of IFNγ responses in subjects with chronic HBV infection who are in immunoregulatory phase or undergoing aggressive treatment. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were tested in an 18-hour IFNγ ELISpot assay with a pool of overlapping peptides representing specific regions of the HBV proteome. Restimulated with one of -23 (5 μg per peptide per mL). FIG. 10 is a graph showing the specificity of IFNγ responses to HBV-derived short peptide pools in HBV-infected subjects grouped by infecting HBV genotype. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were tested in an 18-hour IFNγ ELISpot assay with a pool of overlapping peptides representing specific regions of the HBV proteome. Restimulated with one of -23 (5 μg per peptide per mL). FIG. 10 is a graph showing IFNγ responses to HBV-derived short peptide pools in subjects with chronic HBV infection grouped by ethnic background. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were tested in an 18-hour IFNγ ELISpot assay with a pool of overlapping peptides representing specific regions of the HBV proteome. Restimulated with one of -23 (5 μg per peptide per mL). IFNγ by CD4 and CD8 T cells in PBMC from chronic HBV subjects and PBMC from healthy control subjects after stimulation with HBV polymerase- and core-derived short peptide pools. Representative dot plots of production. Subject-derived PBMCs were stimulated with a short peptide pool library (0.1 μg per peptide per mL) for 10 days followed by HBV-derived short peptide pool 2 or Overnight stimulation with 14 (5 μg per peptide per mL). Results are expressed as a percentage of IFNγ-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. IFNγ against HBV-derived short peptide pools representing 35-40mer peptides in PBMC from healthy subjects and from HBeAg-negative subjects with chronic HBV infection who are in immunoregulatory phase or undergoing active treatment. FIG. 11 is a graph showing a comparison of responses; FIG. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were tested in an 18-hour IFNγ ELISpot assay with duplicates, each representing a 35-40-mer HBV proteome region. Restimulation was with one of the peptide pools 24-46 (5 μg per peptide per mL). FIG. 10 is a graph showing the specificity of IFNγ responses to HBV-derived short peptide pools representing 35-40mer peptides in HBV-infected subjects grouped by infecting HBV genotype. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were analyzed in an 18-hour IFNγ ELISpot assay with duplicates, each representing a specific region of the HBV proteome. Restimulation was with one of pools 24-46 of peptides. FIG. 10 is a graph showing IFNγ responses to HBV-derived short peptide pools representing 35-40mer peptides in HBeAg-negative subjects with chronic HBV infection grouped by ethnic background. After 10 days of culture with an HBV-derived overlapping short peptide pool library (0.1 μg per peptide per mL), PBMCs were tested in an 18-hour IFNγ ELISpot assay with duplicates, each representing a 35-40-mer HBV proteome region. Restimulation was with one of the peptide pools 24-46 (5 μg per peptide per mL). FIG. 10 is a graph depicting a schematic of cytokine responses by PBMC from HBV-infected subjects to individual short peptide pools representing 35-40mer peptides. After 10 days of short-term culture with an HBV-derived short peptide pool library, PBMCs from eAg-negative subjects with chronic HBV infection (n=7-14) were tested in HBV peptide pools 25, 38, 26, 39, Overnight incubation was with one of 42, 43, 28 and 31 (denoting peptides P113, P753, P151, P797, P856, P877, P277 and P376) at a final concentration of 5 μg per peptide per mL. Cells were stained for extracellular expression of CD3, CD4 and CD8 followed by intracellular expression of IFNγ, IL-2 and TNFα. Cells were evaluated by flow cytometry. Cytokine expression was normalized to media negative controls for each subject. Data show mean expression for each cytokine evaluated. The width of the response is indicated above each of the stacked bars. FIG. 2 is a graph showing the number of IFNγ spot-forming cells (mean values) measured in PBMC from chronic HBV infection (either HBeAg-negative inactive carriers or HBeAg-negative subjects undergoing treatment). Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Graph showing the frequency of responders to the IFNγ ELISpot assay in response to HBV peptides measured in PBMC from chronic HBV infection (either HBeAg-negative inactive carriers or HBeAg-negative subjects undergoing treatment). be. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. FIG. 2 is a graph showing the number of IFNγ spot-forming cells (mean values) measured in PBMC from subjects with chronic HBV infection, grouped by infected HBV genotype. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. FIG. 10 is a graph showing the number of IFNγ spot-forming cells (mean values) measured in PBMC from subjects with chronic HBV infection, grouped by ethnic background. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. FIG. 4 is a graph showing the frequencies of cytokine-producing CD4+ and CD8+ T cells in chronic HBV-derived PBMCs after stimulation with HBV-derived peptides. Figure 13A corresponds to the results obtained for a group of individuals infected with HBV genotype A. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Results are expressed as a percentage of cytokine-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. FIG. 4 is a graph showing the frequencies of cytokine-producing CD4+ and CD8+ T cells in chronic HBV-derived PBMCs after stimulation with HBV-derived peptides. Figure 13B corresponds to the results obtained for a group of individuals infected with HBV genotype B. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Results are expressed as a percentage of cytokine-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. FIG. 4 is a graph showing the frequencies of cytokine-producing CD4+ and CD8+ T cells in chronic HBV-derived PBMCs after stimulation with HBV-derived peptides. Figure 13C corresponds to the results obtained for a group of individuals infected with HBV genotype C. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Results are expressed as a percentage of cytokine-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. FIG. 4 is a graph showing the frequencies of cytokine-producing CD4+ and CD8+ T cells in chronic HBV-derived PBMCs after stimulation with HBV-derived peptides. Figure 13D corresponds to the results obtained for a group of individuals infected with HBV genotype D. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Results are expressed as a percentage of cytokine-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. FIG. 4 is a graph showing the frequencies of cytokine-producing CD4+ and CD8+ T cells in chronic HBV-derived PBMCs after stimulation with HBV-derived peptides. Figure 13E corresponds to the results obtained for a group of individuals infected with HBV genotypes other than A/B/C/D. Nine unconjugated HBV peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) (0.1 μg per peptide per mL) ) for 10 days, PBMCs were restimulated with individual peptides at a concentration of 5 μg/ml in an 18 hour IFNγ ELISpot assay. Results are expressed as a percentage of cytokine-producing cells relative to the parental CD3/CD4 or parental CD3/CD8 T cell population. Stimulation with culture medium or PMA/ionomycin was used as negative and positive controls, respectively, and the gating strategy was based on IFNγ production of the negative controls. Graph showing IFNγ production by splenocytes from BALB/c mice (n=7) immunized with FP-02.1 or NP02.1. This graph shows the number of IFNγ spot-forming cells per 10 ⁶ splenocytes measured in response to the nine peptide components of the vaccine. Statistical analysis was performed using the paired t-test. ns = not significant. Graph showing IFNγ production by splenocytes from BALB/c mice (n=7) immunized with FP-02.1 or NP02.1. The graph shows the number of IFNγ spot-forming cells per 10 ⁶ splenocytes measured in response to each of the 9 peptide components of the vaccine. Bars indicate the cumulative median response to each peptide. FP02.1 enhances T cell responses to CTL epitopes restricted by MHC class I molecules after a single immunization. Graph showing IFNγ production by BALB/c mice immunized with FP-02.1 or NP02.1. Number of IFNγ SFCs per 10 ⁶ splenocytes produced in response to a mixture of 9 peptides for each splenocyte population.

Brief Description of the Sequence Listing SEQ ID NOs: 1-38 and SEQ ID NOs: 40-72 are the amino acid sequences of regions of the reference HBV sequence shown in SEQ ID NO: 39 for HBV polymerase shown in Table 1 below.

SEQ ID NO:39 comprises the terminal domain of the polymerase (1-181), the reverse transcriptase domain of the polymerase (182-549), the RNase domain H of the polymerase (550-702), the core protein (703- 914), the X protein (915-1068) and the surface protein (1069-1468) are linearly co-assembled hypothetical HBV protein sequences. Proteome sequences were obtained from a consensus of consensus sequences generated from genotype A, B, C and D consensus sequences.

SEQ ID NOs:73-219 are the amino acid sequences of short peptides in pools 1-46, respectively. SEQ ID NO:220 is the pool 5 amino acid sequence.

Peptide Compositions The present invention provides broadly immunogenic peptide sequences capable of eliciting multiepitopic CD4+ T cell and CD8+ T cell immune responses with broad applicability in terms of population coverage and HBV genotype coverage. A composition is provided comprising: The present invention is a pharmaceutical composition comprising at least one peptide of 15-60 amino acids in length, wherein said peptide comprises a terminal domain of HBV polymerase, a reverse transcriptase domain of HBV polymerase, an RNaseH domain sequence of HBV polymerase. or a fragment of at least 15 consecutive amino acids of HBV core protein. Peptides are selected from peptides that are 15-60 amino acids in length and comprise a sequence of at least 15 contiguous amino acids of one of the sequences shown in SEQ ID NOS: 1-4. The peptide comprises at least one CD8+ T cell epitope and/or at least one CD4+ T cell epitope. Peptides induce responses in peripheral blood mononuclear cells (PBMC) from at least one individual with chronic HBV infection in an in vitro assay.

The composition may comprise multiple peptides having the properties defined above. The composition may be capable of eliciting an immune response in peripheral blood mononuclear cells (PBMC) from at least two individuals of different ethnicity and/or two individuals with different HBV genotypes being infected.

Peptide Sequences Compositions of the invention comprise at least 15 contiguous amino acids from one of SEQ ID NOS: 1-4, such as at least 20, 25, 29, 30, 31, 32, 33, 34 or 35 amino acids. may comprise one or more peptides comprising SEQ ID NOs: 1, 2 and 3 are the HBV polymerase sequences. SEQ ID NO: 4 is the HBV core protein sequence.

These regions are selected such that peptides present in the compositions of the invention may comprise at least 15, 20, 25, 30, 32, 33, 34 or 35 amino acids from one of SEQ ID NOS:5-13. It can be further subdivided. Peptides within these subregions preferably contain sequences within one of SEQ ID NOS: 14-23.

Exemplary short peptides within SEQ ID NOs:1-4 are shown in SEQ ID NOs:80-117 and SEQ ID NOs:142-184. Preferred exemplary short peptides are shown in SEQ ID NOs:80-83, SEQ ID NOs:86-89, SEQ ID NOs:98-101, SEQ ID NOs:105-112, SEQ ID NOs:146-150, SEQ ID NOs:163-166 and SEQ ID NOs:169-181. It is Compositions of the invention may include peptides containing one or more of these short sequences.

Particularly preferred peptides derived from these HBV polymerase sequences include one of the sequences shown in SEQ ID NOs:24-29. SEQ ID NO:24 is a preferred region of SEQ ID NOS:1, 5 and 14. SEQ ID NO:25 is a preferred region of SEQ ID NOs:1, 6 and 15. SEQ ID NO:26 is a preferred region of SEQ ID NOs:2, 7 and 16. SEQ ID NO:27 is a preferred region of SEQ ID NOs:2, 8 and 17. SEQ ID NO:28 is a preferred region of SEQ ID NOs:2, 9 and 18. SEQ ID NO:29 is the preferred region of SEQ ID NOS:3 and 19.

A particularly preferred peptide derived from the above HBV core protein sequence (SEQ ID NO:4) comprises one of the sequences shown in SEQ ID NOS:30-33. SEQ ID NO:30 is a preferred region of SEQ ID NOs:10 and 20. SEQ ID NO:31 is a preferred region of SEQ ID NOs:11 and 21. SEQ ID NO:32 is a preferred region of SEQ ID NOS:12 and 22. SEQ ID NO:33 is a preferred region of SEQ ID NOs:13 and 23.

Other preferred peptides are those contained within the sequences shown in SEQ ID NOS: 24-33, and at least 20, such as 25, 29, 30, 31 within one of these sequences. It includes peptides containing 1, 32, 33 or 34 contiguous amino acids.

The composition may further comprise at least one peptide of 15-60 amino acids in length, wherein said peptide comprises a fragment of at least 15 consecutive amino acids of an HBV surface protein. HBV surface protein peptides are typically 15-60 amino acids in length and are selected from peptides comprising a sequence of at least 15 contiguous amino acids of the sequence shown in SEQ ID NO:55.

The HBV surface protein peptide may comprise at least 15, 20, 25, 30, 32, 33, 34 or 35 amino acids from one of SEQ ID NO:55, preferably SEQ ID NO:71.

Exemplary short peptides within SEQ ID NOs:55 and 71 are shown in SEQ ID NOs:204-210 and 205-209, respectively. Compositions of the invention may include peptides containing one or more of these short sequences.

A particularly preferred peptide derived from these HBV surface protein sequences comprises one of the sequences shown in SEQ ID NO:221. SEQ ID NO:221 is a preferred region of SEQ ID NOS:55 and 71.

Still other peptides that can be included in the compositions of the invention are one or more within one of the sequences set forth in one of SEQ ID NOS: 1-33, 55, 71 and 221, such as: Peptides containing sequences containing 2, 3 or 4 amino acid substitutions, additions or deletions, preferably substitutions. One, two, three or more amino acids within the contiguous sequence may be substituted. Substitutions within the designated sequences include mutations to remove cysteine residues. For example, cysteine residues can be replaced with serine residues.

Typically such peptides are at least 15 or 20, such as 25, 29, 30, 31, within one of SEQ ID NOS: 1-33, 55, 71 and 221, for 32, 33, 34, 35, 36, 37, 38, 39 or 40 contiguous amino acids, or as shown in SEQ ID NOs:24-33 and SEQ ID NO:221 have at least 80%, e.g., at least 85%, 90%, 95%, or 98% sequence identity to the full length of one of the sequences (e.g., National Center for Biotechnology Information (blast.ncbi.nlm determined using the BLAST program available at (.nih.gov/Blast.cgi)). Such peptides include sequences that match amino acid sequences of HBV genotypes A, B, C, D, E or F within the equivalent regions of the HBV polymerase or core protein.

Peptides may include additional sequences provided their total length does not exceed 60 amino acids. For example, the peptide is at least 20 from within one of the sequences set forth in one of SEQ ID NOs: 1-33, 55, 71 and 221, preferably SEQ ID NOs: 24-33 and SEQ ID NO: 221, such as , 25, 29, 30, 31, 32, 33, 34 or 35 contiguous amino acids in length from 15 amino acids, 20 amino acids or 25 amino acids, 30 amino acids, 31 It may be up to amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, 55 amino acids or 60 amino acids.

Thus, peptides are typically 15 or 20 to 60 amino acids in length, eg 25-50 amino acids, preferably 30-40 amino acids, eg 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids. , 35 amino acids, 36 amino acids, 37 amino acids, 38 or 39 amino acids.

A peptide may include additional sequences. Additional sequences can facilitate the manufacture or formulation of the peptide or enhance the stability of the peptide. For example, peptides may be added with one or more additional amino acids, typically at the N-terminus and/or C-terminus, to enhance the net positive charge of the peptide and/or to reduce the hydrophobicity of the peptide. may contain The net positive charge may be increased so that the isoelectric point of the peptide is 7 or higher.

In one aspect of the invention, one or more, for example two or three, positively charged amino acids (arginine and/or lysine) are added to the N-terminus and/or C-terminus of one or more of the peptides in the composition. Append. For example, three lysine residues may be added to the N-terminus and/or C-terminus of one or more of the peptides. Typically adding a positive amino acid to the terminal(s) of a peptide that has an overall hydrophobicity greater than 65%, a net charge less than zero, and/or contains a cluster of hydrophobic amino acids .

Particular examples of peptides containing lysine residues at the N-terminus and/or C-terminus are shown in SEQ ID NOs:34-38 and 222.

Peptides may include one or more epitopes that are not present in the consensus HBV sequence. One such example is the use of fusion peptides, in which a promiscuous T helper epitope is covalently linked to a consensus sequence (optionally via a polypeptide linker or spacer). By way of example, the softly tolerated T helper epitope may be the PADRE peptide, the tetanus toxoid peptide (830-843) or influenza hemagglutinin, HA (307-319).

If the peptide is conjugated to a fluorocarbon, the terminus of the peptide is altered, e.g., the terminus not conjugated with a fluorocarbon or other conjugate, to facilitate solubility of the fluorocarbon-peptide construct, e.g., by forming micelles. can do. N-terminal or C-terminal amino acid residues of the peptide can be modified to facilitate large-scale synthesis of the construct. If the desired peptide is particularly susceptible to cleavage by peptidases, the normal peptide bond can be replaced with a noncleavable peptidomimetic. Such conjugation and synthesis methods are well known in the art.

A peptide may be a native peptide. Peptides may be modified to increase longevity, eg, half-life or persistence at the site of administration of the peptide in vivo, or to target the peptide to antigen-presenting cells. For example, an immunogenic peptide may contain one or more non-naturally occurring amino acids and/or non-naturally occurring covalent bonds to covalently connect neighboring amino acids. In certain embodiments, the non-canonical, non-naturally occurring amino acids do not interfere with the peptide's ability to interact with MHC molecules, even though they remain cross-reactive with T cells that recognize the native sequence. For example, it can be incorporated into an immunogenic peptide. Unnatural amino acids can be used to improve peptide resistance to proteases or chemical stability. Examples of unnatural amino acids include D-amino acids and cysteine modifications.

Peptides may be coupled to carriers such as protein carriers or delivery vectors. Suitable delivery vectors include lipopeptides, eg fatty acyl chains such as monopalmitoyl chains, virosomes, liposomes and cell penetrating peptides such as penetratin, and transactivators of transcription (TAT).

It is preferred that one or more, preferably all, of the HBV peptides in the composition of the invention are covalently linked to a fluorocarbon vector.

Peptide Combinations Compositions of the invention may comprise multiple peptides. Thus, the composition may comprise at least 2, for example at least 3, 4, 5, 6, 7, 8, 9, 10 or more peptides, each of which is described above. comprising a sequence of at least 15 contiguous amino acids of one of SEQ ID NOs: 1-4, as follows. The composition may further comprise a peptide comprising a sequence of at least 15 contiguous amino acids of SEQ ID NO:55 as described above.

In one aspect, the composition comprises at least 15 amino acids of one of the sequences set forth in SEQ ID NOs: 1-3 and at least 15 amino acids of the sequence set forth in SEQ ID NO: 4. and optionally at least one peptide comprising at least 15 amino acids of one of the sequences shown in SEQ ID NO:55. For example, the composition comprises at least 15 amino acids of one of the sequences shown in SEQ ID NOs: 24, 25, 26, 27, 28, 29 and 34 and SEQ ID NOs: 30-33 and 35-38. at least one peptide comprising at least 15 amino acids of one of the sequences of

In another aspect, the composition comprises at least one peptide comprising a sequence of at least 15 contiguous amino acids of one of SEQ ID NOs: 1 and/or 2, as described above, and SEQ ID NOs: 3 or 4, as described above. at least one peptide comprising a sequence of at least 15 contiguous amino acids of For example, the composition comprises a peptide comprising a sequence of at least 15 contiguous amino acids of SEQ ID NO: 1 or 2 as described above (or a peptide comprising both sequences of SEQ ID NOs: 5 and 6) and SEQ ID NO: 10 as described above. Peptides containing at least 15 contiguous amino acids of any one of -13 may be included. The invention includes peptides comprising a sequence of at least 15 contiguous amino acids of any two, three, four, five or all of SEQ ID NOS: 5, 6, 7, 8 and 9 as described above. may and/or include peptides comprising a sequence of at least 15 contiguous amino acids of any two, three or all of SEQ ID NOS: 10-13 as described above.

A peptide present in a composition of the invention may consist of, or consist essentially of, one of the sequences shown in SEQ ID NOs:24-38. The HBV surface protein peptides present in the compositions of the invention may consist or consist essentially of one of the sequences shown in SEQ ID NOs:221 and 222. Accordingly, the present invention provides at least one peptide consisting of, consisting essentially of, or comprising the amino acid sequence shown in one of SEQ ID NOS: 24-38, such as two or more such peptides. A pharmaceutical composition is provided comprising a peptide, and optionally at least one peptide consisting of, consisting essentially of, or comprising the amino acid sequence shown in SEQ ID NO: 221 or 222. The composition comprises at least 2, such as 3, 4, 5, 6, 7, 8, 9 or 10, comprising the sequences set forth in SEQ ID NOS: 24-33 and SEQ ID NO: 221. It may contain peptides. In one embodiment, one or more of the peptides comprising one of SEQ ID NOS:24-33 and SEQ ID NO:221 may include a lysine residue at the N-terminus or C-terminus. More specifically, peptides comprising SEQ ID NOs:26, 29, 30, 31, 32 and 221 may have the sequences shown in SEQ ID NOs:34-38 and SEQ ID NO:222, respectively.

For example, the composition comprises at least one of the peptides comprising, consisting of, or consisting essentially of the sequences set forth in SEQ ID NOs: 24, 25, 27, 28, 33, 34, 35, 36, 37 and 38. 2, such as 3, 4, 5, 6, 7, 8, 9 or 10 or including SEQ ID NOs: 24, 25, 28, 33, 34, 36, 37 and 38 , at least 2, for example 3, 4, 5, 6, 7 or 8, of peptides consisting of or consisting essentially of. Any possible combination of these peptides may be present in the composition of the invention. Preferred combinations include one or more of SEQ ID NOS:24, 25, 28, 33 and 31/37, such as any two, any three, any four or all, preferably SEQ ID NO:24 and / or 25. For example, the combination of SEQ ID NO:24 and SEQ ID NO:33 results in a composition containing epitopes that bind to 7 class I alleles and 7 class II alleles. The composition may further comprise a peptide comprising, consisting of, or consisting essentially of SEQ ID NO:221 or 222.

For example, the composition comprises eight peptides comprising the following sequences: SEQ ID NOS: 24, 25, 26, 28, 30, 31, 32 and 33, and optionally a ninth peptide comprising SEQ ID NO: 222. good.

One of the peptides, such as a peptide comprising SEQ ID NO:28, can be replaced with a peptide comprising SEQ ID NO:27 and/or one peptide can be replaced with a peptide comprising SEQ ID NO:29. . One or more of the peptides are at least 80% over their entire length relative to the shorter peptides described above, e.g., peptides having at least 20 contiguous amino acids of the peptide to be replaced, or the amino acid sequence of the peptide to be replaced. can be substituted with a peptide having the identity of

Preferably, the composition comprises at least one peptide derived from the HBV polymerase described above, more preferably from the terminal domain of HBV polymerase. In particularly preferred embodiments, the HBV polymerase peptide comprises at least one amino acid sequence within SEQ ID NO:1, such as at least one sequence within SEQ ID NO:5, 6, 14, 15, 24 or 25. For example, such peptides may comprise the amino acid sequence shown in one of SEQ ID NOs:80, 81, 82, 83, 86, 87, 88, 89, 24 or 25.

HBV genotypes The combination of peptide sequences in the composition results in epitopes that are present in multiple HBV genotypes, preferably both CD8+ and CD4+ epitopes. HBV genotypes include genotypes A, B, C, D, E and F. For example, long peptides may contain epitopes from at least two HBV genotypes, such as A and D (the most prevalent genotype in Europe) or B and C (the most prevalent genotype in Asia). may contain More preferably, the composition comprises epitopes from at least three HBV genotypes, such as A, B and C, A, B and D, A, C and D, or B, C and D, or the like. Most preferably, the composition comprises epitopes from at least HBV genotypes A, B, C and D. In addition to comprising any combination of epitopes from any combination of one or more of genotypes A, B, C and D, the composition may contain epitopes for genotypes E, F and/or G. may contain. This can be determined by any suitable means, such as by using the in vitro PBMC assays described herein.

Accordingly, the present invention provides an individual infected with HBV genotype A, an individual infected with HBV genotype B, an individual infected with HBV genotype C, an individual infected with HBV genotype D and Compositions are provided that are capable of inducing an immune response in PBMC from 2, 3, 4, or all of the individuals infected with another HBV genotype.

two of an individual infected with HBV genotype A, an individual infected with HBV genotype B, an individual infected with HBV genotype C, and an individual infected with HBV genotype D; A composition of the invention capable of inducing an immune response in three individuals or in all individuals may comprise at least one peptide selected from at least two, preferably three or all of the following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 67, 22 or 23;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 17, 18, 21 or 67;
(iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 16, 60, 19, 20 or 22;
(iv) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 67, 22 or 23;

The composition may further comprise a peptide comprising at least 15 contiguous amino acids of SEQ ID NO:55 or SEQ ID NO:71.

For example, such compositions may comprise peptides selected from at least two, preferably three or all of the following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 20, 21, 67, 22 or 23;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 17 or 18;
(iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 16, 60 or 19; and
(iv) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14 or 15;

The composition may further comprise a peptide comprising at least 15 contiguous amino acids of SEQ ID NO:55 or SEQ ID NO:71.

Suitable peptides comprising at least 15 amino acids of the specified sequences are described in more detail herein and include, in particular, the peptides of SEQ ID NOs: 24-38 listed in Table 4, as well as the sequences Peptides numbered 221 and 222 are included.

In one aspect, the composition of the present invention comprises at least one individual chronically infected with HBV genotype A, one chronically infected with HBV genotype B, and one chronically infected with HBV genotype C. It elicits an in vitro response in peripheral blood mononuclear cells (PBMC) from one infected and one chronically infected HBV genotype D individual. This can be determined by any suitable method, such as those described in the Examples herein. Individuals may be of the same or different ethnicity, preferably of at least two different ethnicities. The HLA subtypes of the individual may be the same or different, preferably at least two different HLA subtypes.

Ethnicity The present invention provides compositions capable of eliciting an immune response in individuals of at least two, eg, three or more, different ethnicities. This can be assessed using the in vitro PBMC assay described in the Examples. The compositions of the present invention may be used in two individuals: an HBV-infected Asian or Indian individual, an HBV-infected Caucasian individual, and an HBV-infected African or Arab individual. , elicit an immune response in PBMC from 3 individuals or from all individuals.

in 2, 3 or all HBV-infected Asian or Indian individuals, HBV-infected Caucasian individuals, and HBV-infected African or Arab individuals A composition of the invention capable of inducing an immune response may comprise at least one peptide selected from at least two, preferably three or all of the following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 16, 60, 17, 18, 20, 21, 67 or 22;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 19, 20, 22 or 23;
(iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 67, 22 or 23;

The composition may further comprise a peptide comprising at least 15 contiguous amino acids of SEQ ID NO:55 or SEQ ID NO:71.

For example, such compositions may comprise peptides selected from at least two, preferably three or all of the following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 16, 60, 17, 18;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15 or 19; and (iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 22 or 23. .

The composition may further comprise a peptide comprising at least 15 contiguous amino acids of SEQ ID NO:55 or SEQ ID NO:71.

Suitable peptides comprising at least 15 amino acids of the specified sequences are described in more detail herein and include, in particular, the peptides of SEQ ID NOs: 24-38 listed in Table 4, as well as the sequences Peptides numbered 221 and 222 are included.

epitope
HLA class I and HLA class II molecules are polymorphic and their frequency varies between ethnic groups. The majority of the polymorphisms are located within the peptide binding region, and as a result each variant is believed to bind a unique repertoire of peptide ligands. HLA polymorphisms represent a major problem for vaccine designers, as they underlie differential peptide binding. Moreover, certain HLA alleles are expressed at dramatically different frequencies in different ethnicities.

Despite such polymorphisms, HLA molecules bind to overlapping sets of peptides and can therefore be grouped into supertypes accordingly (Lund et al. (2004) Immunogenetics 55(12):797 -810, Sette et al. (1999) Immunogenetics 50(3-4):201-212). A supertype is defined as a family of distinct HLA molecules that have similar peptide binding repertoires and thus share an overlapping set of peptides. In other words, peptides that bind to HLA alleles belonging to a given supertype are likely to exhibit binding activity to other supertype members.

The ability of peptides to bind to different HLA class II alleles has been described in Texier et al. (2000) J Immunol 164:3177-3184, Texier et al. (2001) Eur J Immunol 31:1837-1846 and Castelli et al. (2002) J Immunol 169:6928. -6934, using a heterologous competition assay using specific biotinylated tracer peptides for each HLA class II allele.

The following 9 HLA class II alleles were sequenced and binding motif specific as described in Lund et al. (2004) Immunogenetics 55(12):797-810 and Greenbaum et al. Major supertypes or HLA clusters based on sex are indicated: HLA-DR1 (α1*01:01; β1*01:01), HLA-DR3 (α1*01:01; β1*01:01), HLA- DR4 (α1*01:01; β1*04:01), HLA-DR7 (α1*01:01; β1*07:01), HLA-DR11 (α1*01:01; β1*11:01), HLA -DR13 (α1*01:01; β1*13:01), HLA-DR15 (α1*01:01; β1*15:01), HLA-DR51 (α1*01:01; β5*01:01) and HLA-DP4 (α1*01:03; β1*04:01). These alleles have a high distribution across different ethnicities (see Wilson et al. (2001) J Virol. 75(9):4195-4207).

Peptides present in the compositions of the invention are typically of at least 2, preferably at least 3, of the 9 major HLA class II alleles, e.g. binds at least two, preferably at least three, of the HLA class II alleles of one or more of the peptides present in the composition are at least 4, 5, 6, 7, 8 or all of the 9 major HLA class II alleles; can bind at least 4, 5, 6 or all of the 7 HLA class II alleles described in . Compositions of the invention may target at least 7, at least 8 or all 9 of the above major HLA class II alleles, e.g. It is preferred to include a peptide that can bind.

The number of HLA class I binding registers contained in each peptide can be determined by determining the peptide's ability to bind to various frequently occurring HLA class I molecules. HLA class I binding may be measured using the ProImmune REVEAL® MHC-peptide Binding Assay (ProImmune Ltd, Oxford, UK). HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*2402, HLA-B*0702, representing the major HLA class I supertypes by REVEAL™ MHC-peptide binding assay. The ability of each peptide to stabilize ternary MHC-peptide complexes for HLA-B*0801, HLA-B*3501 is measured. Each peptide tested is given a score against the pass/fail control peptide and compared to the positive control peptide.

HLA class I molecules bind short peptides that vary in length from 8 to 11 amino acids. Theoretically, 102 short peptides (27×8mers, 26×9mers, 25×10mers and 24×11mers) can be derived from a 35mer peptide sequence. To limit the number of peptides tested, only nonamer peptides (the most frequent length for HLA class I binding peptides) with good prediction scores based on publicly available algorithms were used. Binding assays can be performed.

The following HLA class I alleles are highly represented in the human population, (2) they belong to well-defined HLA supertypes (http://bioinformatics.nmdp.org/): HLA-A* 0101, HLA-A*0201, HLA-A*0301, HLA-A*2402, HLA-B*0702, HLA-B*0801, HLA-B*3501 and HLA-A*1101.

A peptide present in a composition of the invention will typically have at least one, preferably at least two or at least three of these HLA class I alleles, e.g. preferably the 7 HLA class I alleles described in Example 9. One or more of the peptides present in the composition may comprise short peptides that bind at least 4, 5, 6 or all of the 7 HLA class I alleles. Compositions of the invention preferably comprise peptides, including short peptides, capable of binding at least 5, at least 6, or all 7 of the above HLA class I alleles.

Pharmaceutical compositions of the invention typically comprise one or more peptides comprising one or more T cell epitopes that bind to different MHC alleles to provide broad population coverage. Compositions may comprise peptides known or predicted to contain one or more MHC binding motifs associated with frequent MHC alleles within a particular ethnic group or across multiple ethnic groups. . The composition may comprise one or more CD4+ T cell epitopes and CD8+ T cell epitopes with flexible tolerance that bind to more than one allelic variant. The combination of peptide sequences in the composition provides T cell epitopes that bind to different HLA subtypes.

In one aspect, the compositions of the invention elicit a response in vitro in peripheral blood mononuclear cells (PBMC) from at least two individuals with different HLA subtypes. The composition is capable of eliciting an immune response in at least 3, 4, 5, 6 or 7 individuals, each having a different HLA genotype and which may be of different ethnicities.

Fluorocarbons Fluorocarbons may contain one or more chains derived from perfluorocarbon or mixed fluorocarbon/hydrocarbon groups, may be saturated or unsaturated, and each chain may contain from 3 to 30 carbons. have atoms. Thus, the chain at the fluorocarbon linkages is typically saturated or unsaturated, preferably saturated. The chain in the fluorocarbon bond may be linear or branched, preferably linear. Each chain typically has 3 to 30 carbon atoms, 5 to 25 carbon atoms, or 8 to 20 carbon atoms. To covalently link the fluorocarbon vector and the peptide, a reactive group or ligand such as -CO-, -NH-, S, O or any other suitable group is included in the vector. The use of such ligands to achieve covalent linkage is well known in the art. The reactive group may be located anywhere on the fluorocarbon vector.

Coupling of fluorocarbon vectors and peptides may be accomplished through functional groups such as -OH, -SH, -COOH and _-NH2 that are naturally present or introduced at any site of the peptide. Examples of such linkages include amide bonds, hydrazone bonds, disulfide bonds, thioether bonds and oxime bonds.

Optionally, a spacer element (peptidic or non-peptidic) can be incorporated to allow cleavage of the peptide from the fluorocarbon element for optimization of processing and steric presentation of the peptide within antigen-presenting cells. . Spacers can also be incorporated to aid in the synthesis of the molecule and to improve its stability and/or solubility. Examples of spacers include polyethylene glycol (PEG), or amino acids such as lysine or arginine that can be cleaved by proteolytic enzymes.

In one embodiment, the _fluorocarbon -linked peptide may have the chemical structure _CmFn - _CyHx- (Sp)-R or derivatives thereof, where m=3-30 _and n ≦2m+1, y=0-15, x≦2y, (m+y)=3-30, Sp is an optional chemical spacer moiety, R is immunogenic is a peptide. Typically, m and n satisfy the relationship 2m-1≤n≤2m+1, preferably n=2m+1. Typically, x and y satisfy the relationship 2y-2≤x≤2y, preferably x=2y. Preferably, _{the CmFn - CyHx} moiety is linear.

m is preferably 5-15, more preferably 8-12. Similarly, y is preferably 0-8, more preferably 0-6 or 0-4. C _m F _n -C _y H _x moieties are saturated (i.e. n=2m+1 and x=2y), linear and m=8-12 and y=0-6 or 0 ~4 is preferred.

の2H, 2H, 3H, 3H-ペルフルオロウンデカン酸に由来する。 In a particular example, the fluorocarbon vector has the formula:

derived from 2H, 2H, 3H, 3H-perfluoroundecanoic acid in

A preferred _fluorocarbon linkage is therefore a linear saturated moiety _C8F17 ( _CH2 ) _2- derived from _{C8F17 ( CH2} ) _2COOH .

Further examples _of fluorocarbon linkages _{are C6F13} ( _CH2 ) _2COOH , _C7F15 ( _CH2 ) _{2COOH , C9F19} ( _CH2 ) _2COOH , _C10F21 ( _CH2 ) _2, respectively _. COOH _{, C5F11 ( CH2 ) 3COOH , C6F13 (CH2)3COOH, C7F15 ( CH2 ) 3COOH} , _{C8F17 ( CH2} ) _3COOH and _C9F19 derived from ( _{CH2 ) 3COOH} : _C6F13 ( _{CH2 ) 2-} , _C7F15 ( _CH2 ) _2- , _{C9F19 ( CH2} ) _{2- , C10F21} ( _CH2 ) _{2- , C5F11} ( _{CH2 ) 3-} , _C6F13 ( _{CH2 ) 3- , C7F15} ( _CH2 ) _3- , _C8F17 ( _CH2 ) _3- and _C9F19 ( _CH2 ) _{3- .}

を有し、式中、Sp及びRは上で定義された通りである。ある特定の実施形態では、Spはリシン残基に由来し、式-CONH-(CH₂)₄-CH(NH₂)-CO-を有する。Rは配列番号1～14のうちの任意の1つであることが好ましく、Rは配列番号1～6のうちの任意の1つであることが好ましい。各ペプチド、例えば、配列番号1、2、3、4、5又は6のN末端アミノ酸のアミノ基は、式-CONH-(CH₂)₄-CH(NH₂)-CO-のスペーサーのC末端カルボキシ基とアミド結合を形成する。 A preferred example of a suitable structure for a fluorocarbon vector-antigen construct is the formula:

wherein Sp and R are as defined above. In certain embodiments, Sp is derived from a lysine residue and has the formula -CONH-( _CH2 ) ₄ -CH( _NH2 )-CO-. Preferably, R is any one of SEQ ID NOs: 1-14, preferably R is any one of SEQ ID NOs: 1-6. _The amino group of the N-terminal amino acid _{of each} peptide, e.g. Forms an amide bond with a carboxyl group.

For the present invention, the fluorocarbon linkage may be modified such that the resulting compound is still capable of delivering peptides to antigen presenting cells. Thus, for example, some fluorine atoms may be replaced by other halogen atoms such as chlorine, bromine or iodine. Additionally, it is possible to replace some of the fluorine atoms with methyl groups and still retain the properties of the molecules described herein.

Peptides may be linked to the fluorocarbon vector via a spacer moiety. Preferably, the spacer moiety is a lysine residue. This spacer residue may be present in addition to any terminal lysine residues described above, so a peptide may have, for example, a total of four N-terminal lysine residues. Accordingly, preferred formulations of the invention may comprise a fluorocarbon-linked peptide, wherein the peptide has a C-terminal or N-terminal lysine residue, preferably an N-terminal lysine residue. A terminal lysine within _the peptide is preferably linked to a fluorocarbon having the formula _C8F17 ( _CH2 ) _2COOH . Preferably, the fluorocarbon is coupled to the epsilon chain of the N-terminal lysine residue.

The pharmaceutical compositions described herein optionally include at least 1, 2, 3, 4, 5, 6, 7, 8, each covalently linked to its own fluorocarbon vector. , is intended to include 9 or 10 or more immunogenic peptides.

Peptides The invention also provides peptides useful in the compositions of the invention. The peptide may be any one of the above peptides. In particular, the present invention provides one of the sequences shown in SEQ ID NOS:24-33 and SEQ ID NO:221, or one of the sequences shown in SEQ ID NOS:24-33 and SEQ ID NO:221 and at least 80 Peptides up to 40, 50 or 60 amino acids in length are provided that contain sequences that are % identical, eg, at least 85%, 90%, 95% or 98% identical. The peptide may contain additional amino acids as described above. In one particular embodiment, the invention provides a peptide having the sequence shown in one of SEQ ID NOS:34-38 and SEQ ID NO:222. Particularly preferred peptides of the invention comprise, consist essentially of, or consist of the sequences shown in SEQ ID NOS: 24, 25, 28, 30, 31, 32, 33, 34, 36, 37 and 38.

The present invention also provides highly conserved immunogenic peptides derived from the terminal domain of HBV polymerase. These peptides may be any of the HBV polymerase peptides described with respect to the compositions of the invention above. Such peptides are typically 15-60 amino acids in length, contain at least 15 consecutive amino acids of SEQ ID NO: 1 or 2, and are derived from at least one individual chronically infected with HBV. elicits an immune response in vitro in PBMC.

Peptides may be coupled to carriers as described above. In one preferred embodiment, the peptides of the invention are covalently linked to a fluorocarbon vector. Fluorocarbon vectors may be as described above.

Other Components Compositions of the invention may include additional immunogens. The immunogen may be a B cell antigen. B cell antigens can serve to stimulate antibody responses to HBV. A pharmaceutical composition of the invention may comprise, for example, one or more fluorocarbon-linked peptides capable of stimulating a T-cell response and a B-cell antigen.

Suitable immunogens that serve as B cell antigens include protein antigens such as hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg or HBeAg).

In one aspect, the invention provides compositions comprising two or more peptides, such as fluorocarbon-linked peptides, further comprising an adjuvant and/or optionally a pharmaceutically acceptable carrier or excipient. Excipients may be stabilizers or bulking agents necessary for efficient lyophilization. Examples include sorbitol, mannitol, polyvinylpyrrolidone, and mixtures thereof, preferably mannitol. Other excipients that may be present include preservatives such as antioxidants, lubricants, cryopreservatives and binders, which are well known in the art.

An adjuvant is an agent capable of modulating the immune response directed against a co-administered antigen with little, if any, direct effect when given by itself. Such adjuvants may enhance the immune response in terms of magnitude and/or cytokine profile. Examples of adjuvants include Freund's adjuvant and its derivatives, muramyl dipeptide (MDP) derivatives, CpG, refinements of natural components of bacteria derived naturally or synthetically such as monophosphoryl lipid A; saponins, aluminum. other known adjuvants or enhancers such as salts and cytokines; oil-in-water adjuvants, water-in-oil adjuvants, immunostimulatory complexes (ISCOMs), liposomes, formulated nanoparticles and microparticles; bacterial toxins and toxoids; , especially gamma-inulin; and TLR agonists.

Preferably, the adjuvant is peptidoglycan (e.g. TDM, MDP, muramyl dipeptide, Murabutide), alum water (e.g. aluminum hydroxide, ADJUMER™ (polyphosphazene) or aluminum phosphate gel), glucan, algammulin, surfactants (e.g. squalane, Tween 80, Pluronics or squalene), calcium phosphate gels, bacterial toxins or toxoids (e.g. cholera holotoxin, cholera toxin-A1-protein A-D-fragment fusion proteins, Cholera toxin subunit B, or block copolymer), liposomes containing cytokines, water-in-oil adjuvant (e.g. Freund's complete adjuvant, Freund's incomplete adjuvant or Montanide such as ISA 51 or ISA 720), water oil adjuvants (e.g. MF-59), inulin-based adjuvants, cytokines (e.g. interferon-gamma, interleukin-1 beta, interleukin-2, interleukin-7 or interleukin-12), ISCOMs (e.g. matrices (iscomatrix), microspheres and microparticles of any composition, and Toll-like receptor agonists (e.g., CpG, ligand of human TLR 1-10, ligand of mouse TLR 1-13, ISS-1018, IC31, imidazo quinoline, poly(I:C), monophosphoryl lipid A, Ribi529, cholera toxin, thermolabile toxin, Pam3Cys or flagellin).

Preparation of Pharmaceutical Compositions Pharmaceutical compositions of the present invention are prepared by solubilizing at least one peptide, such as a fluorocarbon-linked peptide, in acetic acid or other solvent as a first step in formulating a pharmaceutical product. can be prepared. Examples of other solvents that can be used to disperse one or more of the fluorocarbon-linked peptides in the blend include phosphate buffered saline (PBS), propan-2-ol, tert-butanol, acetone, and Other organic solvents are included. Techniques for solubilizing fluorocarbon vector-peptide conjugates are described in WO2012/090002.

The peptides or fluorocarbon-linked peptides used as starting materials are typically dry. Peptides including peptides shorter than 20 amino acids and/or having less than 50% hydrophobic residues and fluorocarbon-linked peptides can be solubilized in solvents other than acetic acid. Acetic acid is typically used when the peptide has more than 20 amino acids and/or more than 50% hydrophobic residues.

The concentration of the fluorocarbon-linked peptide in solution is typically about 0.1 mM to about 10 mM, such as about 0.5 mM, 1 mM, 2 mM, 2.5 mM or 5 mM. An example of a suitable concentration is about 10 mg/mL.

The input ingredients can be uniformly blended to the desired ratio with any aggregates dispersed, sterilized, and provided in a form suitable for administration. Such examples may include introducing vortexing and/or sonication at a post-blending or post-dilution stage to facilitate solubilization. Other permutations of the manufacturing process flow can include performing sterile filtration earlier in the process, or omitting lyophilization to allow for final presentation in liquid form. .

When different peptides or fluorocarbon-linked peptides are solubilized separately, for example in different solvents or in different concentrations of acetic acid, the solubilized peptides or fluorocarbon-linked peptides are blended to form a peptide or fluorocarbon-linked peptide. to create a mixture of

An optional adjuvant and/or one or more pharmaceutically acceptable excipients can also be added to the solubilized peptide/fluorocarbon-linked peptide or mixture of peptide/fluorocarbon-linked peptide. Typically, solubilized fluorocarbon-linked peptides are mixed with excipients and/or adjuvants.

After solubilization and blending, the solution of fluorocarbon-linked peptide(s) may be diluted. For example, the blend may be diluted in water.

It is preferred to sterilize the solution containing the peptide or fluorocarbon-linked peptide. Sterilization is particularly preferred when the formulation is for systemic use. Any suitable means of sterilization may be used, such as UV sterilization or filter sterilization. It is preferred to use filter sterilization. Sterile filtration may involve a 0.45 μm filter followed by a series of 0.22 μm sterilizing grade filters.

Sterilization may be performed before or after adding any excipients and/or adjuvants.

The compositions of the invention may be in dried form, such as lyophilized form. A composition of the invention may be an aqueous solution, eg, an aqueous solution formed by dissolving a lyophilisate or other dry formulation in an aqueous medium. The aqueous solution is typically at neutral pH.

Drying the formulation facilitates long-term storage. Any suitable drying method may be used. Lyophilization is preferred, but other suitable drying methods such as vacuum drying, spray drying, spray freeze drying or fluid bed drying may be used. The drying procedure may result in the formation of an amorphous cake incorporating the peptide or fluorocarbon-linked peptide.

The sterile composition may be lyophilized for long-term storage. Lyophilization can be achieved by freeze-drying. Freeze-drying typically involves freezing and then drying. For example, a fluorocarbon-linked peptide mixture may be frozen at -80°C for 2 hours and freeze-dried in a freeze-drying apparatus for 24 hours.

A pharmaceutically acceptable composition of the invention may be a solid composition. A fluorocarbon-linked peptide composition may be obtained in the form of a dry powder. The cake resulting from freeze-drying can be crushed into a powder form. Accordingly, the solid composition according to the invention may take the form of free-flowing particles. Solid compositions are typically provided as powders in sealed vials, ampoules or syringes. For inhalation, the powder can be provided in a dry powder inhaler. Alternatively, a solid matrix can be provided as a patch. The powder may be compressed into tablet form.

Dry, eg, lyophilized, peptide or fluorocarbon-linked peptide compositions may be reconstituted prior to administration. As used herein, the term "reconstitution" is understood to mean reconstitution of the dried vaccine product prior to use. After drying, such as lyophilization, the immunogenic peptide, eg, fluorocarbon-linked peptide product, is preferably reconstituted to form a homogeneous isotonic, pH-neutral suspension. Formulations are typically made with water by adding, for example, water for injection, histidine buffer solution (e.g., 28 mM L-histidine buffer), sodium bicarbonate, Tris-HCl, or phosphate buffered saline (PBS). Reconstituted during the phase. Reconstituted formulations are typically dispensed into sterile containers such as vials, syringes, or any other format suitable for storage or administration.

The composition may be stored in a container such as a sterile vial or syringe prior to use.

Medical Uses The present invention provides compositions of the invention for use in the treatment of the human or animal body by therapy. In particular, compositions of the invention are provided for use in methods of treating or preventing HBV infection. The compositions of the invention elicit an immune response that may also be useful in HBV prophylaxis. The compositions of the invention are preferred for use as therapeutic vaccines to treat individuals infected with HBV. The compositions of the invention are particularly useful in treating patients with persistent chronic HBV infection, but can also be used to treat immunotolerant patients or inactive chronic carriers.

The present invention provides therapeutic vaccines as a disruptive technology for treating chronic HBV (CHB). The compositions of the invention enhance antiviral T cell responses and provide spontaneous immune control of HBV infection. This would allow antiviral NUC therapy to be discontinued and could potentially also lead to serological cure of HBV infection. HBsAg decline is used as a predictor of improved long-term clinical outcome. HBsAg levels can be correlated with the number of hepatocytes infected with HBV and are determined by the transcriptional activity of intrahepatic cccDNA that is regulated by various cytokines. Treatment with the compositions of the invention can result in HBsAg loss or HBsAg seroconversion.

The peptides and compositions of the invention are particularly useful in treating CHB patients who have undergone NUC therapy. The peptide also represents an available treatment for HBeAg-positive patients in developing countries who do not have access to long-term NUC therapy. Vaccination of HBV-DNA-suppressed, HBeAg-negative patients, particularly NUC-treated, with the peptide compositions of the invention facilitates and accelerates HBsAg clearance. HBeAg positive patients can also be treated. The compositions of the invention may also be used to treat inactive carriers of HBV.

Hepatitis B virus (HBV) infection is a leading cause of liver-related morbidity and mortality. Compositions of the invention are provided for use in the treatment of liver failure, end-stage liver disease and hepatocellular carcinoma.

The compositions of the invention are useful in the vaccination of patients with hepatitis delta (HDV), the most severe form of viral hepatitis, for which no approved therapy is available and occurs only as a co-infection in HBsAg-positive individuals. be.

The present invention relates to the manufacture of a medicament for treating or preventing HBV infection, particularly CHB, for treating or preventing liver failure, end-stage liver disease or hepatocellular carcinoma, or for treating or preventing HDV. Uses of the pharmaceutical compositions of the invention are also provided.

Similarly, the invention provides a method of treating or preventing HBV infection in a subject in need thereof, comprising administering to said subject a prophylactic or therapeutic amount of a composition of the invention, provide a way.

Compositions of the invention may also be administered in combination with a second therapeutic or prophylactic agent. For example, the second agent may be a further immunogen to further stimulate an immune response, e.g., to stimulate a humoral immune response when the fluorocarbon-linked peptide stimulates a cell-mediated immune response to HBV. (eg, globular antigens or recombinant or naturally occurring antigens). It is understood that the second agent may be a B cell antigen. Suitable B cell antigens include HBsAg, HBcAg and HBeAg.

In preferred embodiments, the second agent is an agent that is known for use in existing HBV therapeutic treatments. Existing HBV therapeutics may be interferons, such as interferon-alpha, or NUCs, such as entecavir and tenofovir. HBV therapeutic treatments may be treatments that block suppressive cell types. Agents useful in such blocking therapy include anti-PD1 blocking antibodies, anti-PD1L blocking antibodies, anti-LAG3 blocking antibodies, anti-TIM3 blocking antibodies, anti-CTLA4 blocking antibodies and cyclophosphamide.

When a second therapeutic or prophylactic agent is used concurrently with the compositions of this invention, administration can be simultaneous or separated. A composition of the invention may be administered before, along with, or after the second therapeutic agent.

The compositions of the invention can be administered to human or animal subjects in vivo using a variety of known routes and techniques. For example, the compositions may be provided as injectable solutions, suspensions, or emulsions and injected parenterally, subcutaneously, orally, epidermally, intradermally, intramuscularly, intraarterially, intraperitoneally, or intravenously using conventional needles and syringes. or may be administered using a liquid jet injection system. The composition may be administered topically to skin or mucosal tissue, for example, nasally, intratracheally, intestinally, sublingually, rectally or vaginally, respiratory administration or pulmonary administration. It may also be provided as a finely divided spray suitable for internal administration. In preferred embodiments, the composition is administered intramuscularly.

Such compositions can be administered to a subject in an amount compatible with the dosage composition, and in an amount that will be prophylactically and/or therapeutically effective. Administration of the compositions of the invention may be either for "prophylaxis" or for "treatment." As used herein, the term "therapeutic" or "treatment" includes any one or more of the following: prevention of infection or reinfection, reduction or elimination of symptoms, and reduction or complete elimination of pathogens. Treatment may be carried out prophylactically (before infection) or therapeutically (after infection).

The choice of carrier, if any, will often depend on the route of delivery of the composition. Within the scope of the present invention, compositions may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents include oral, ocular, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral administration. (including subcutaneous, intramuscular, intravenous, intradermal and transdermal).

The composition may be administered in any suitable form, for example liquid, solid or as an aerosol. For example, oral formulations may take the form of emulsions, syrups or liquids or tablets or capsules, which may be enterically coated to protect the active ingredient from degradation in the stomach. Nasal formulations may be sprays or solutions. Transdermal formulations may be suitable for their particular delivery system and may include patches. Formulations for injection may be solutions or suspensions in distilled water or another pharmaceutically acceptable solvent or suspending agent.

The appropriate dose of prophylactic or therapeutic vaccine to administer to a patient is determined in the clinic. As a guide, however, a suitable human dose may depend on the preferred route of administration and may be 1-1000 μg, eg about 100 μg, 200 μg or 500 μg. Multiple doses may be required to achieve an immunological or clinical effect and, if required, are typically given 2-12 weeks apart. Repeat doses spaced from one month to five years apart may be applied if long-term boosting of the immune response is required.

The following examples are illustrative of the invention.

[Example 1]
Evaluation of ex vivo immunogenicity of HBV-derived short peptide pools in human PBMCs.
Subjects with HBV infection Ninety-nine subjects with clinically defined chronic HBV infection were recruited to the Imperial Healthcare NHS Trust, Chelsea and Westminster Hospital NHS Foundation Trust, and Barts and the London NHS Trust in London. Enrolled in a protocol approved by the REC. After obtaining written informed consent from all subjects, fresh venous blood was collected and PBMCs and plasma were isolated and cryopreserved within 18 hours of blood collection. These subjects met the following criteria: good overall health, HBV-specific therapy: antiviral nucleoside (nucleotide) analog inhibitors and/or interferon therapy (clinically indicated). clinical status (chronic HBV infection, HBeAg-negative, and normal, persistent or intermittent elevation of ALT), HIV-negative, HCV-negative and HDV-negative.

healthy control subject
Cryopreserved PBMCs from 17 subjects were obtained from CTL Technologies. These subjects met the following criteria: good overall health, no vaccination against HBV, HBV surface antigen negative, HBV core antibody negative, HIV negative and HCV negative.

Short-Term Culture of PBMC One vial of PBMC from each subject (containing 1×10 ⁷ cells) was thawed and lymphocyte counts were determined using a Scepter™ handheld automated cell counter. PBMC were cultured in 24-well cell culture plates at a concentration of 1×10 ⁶ cells per mL in 2 mL of culture medium (CM: RPMI-1640 Glutamax supplemented with 5% human AB serum) for a total of 11 days. Cells were treated with 1 mL of a peptide pool containing 144 overlapping HBV-derived short peptides (SEQ ID NOs:73-210 and SEQ ID NOs:214-219) ranging in length from 15-20 amino acids and overlapping by 10-13 amino acids. A final concentration of 0.1 μg per peptide was used for stimulation. On day 4, IL-2 and IL-15 were added to the cultures to final concentrations of 10 IU/mL and 10 ng/mL, respectively. On day 10, cells were washed twice in CM and cultured for an additional day with 10 IU/mL IL-2. On day 11, cells were washed twice in CM, counted and incorporated into human IFNγ ELISpot assays or intracellular cytokine staining.

Human IFNγ ELISpot assay
96-well multiscreen PVDF filter plates (Millipore) were coated with 100 μl (1:80) of anti-human IFNγ capture mAb (R&D Systems) overnight at 4°C. Plates were then blocked with PBS supplemented with 1% BSA and 5% sucrose for 2 hours at 4°C. Cells were plated at 5×10 ⁴ PBMCs per well in triplicate wells. The final concentration of antigen used was 22 short peptide pools from HBV (see below, which could not be prepared as peptides of SEQ ID NOs: 212 and 213. Pool 22 could not be dispersed due to insolubility). ) and HIV-3 35mer negative peptide controls: 5 μg per peptide per mL, PHA positive controls: 1 μg/mL. ELISpot plates were incubated for 18 hours at 37° C., 5% CO ₂ in a humidified environment. Plates were then washed and incubated with 100 μl (1:80) biotinylated anti-human IFNγ detection mAb (R&D Systems) for 2 hours at room temperature. After washing, plates were incubated with streptavidin-conjugated alkaline phosphatase (1:80) for 1 hour followed by substrate (30 minutes) according to the manufacturer's instructions (R&D Systems). Generated spots were counted using an automated plate counting system (CTL Europe).

Intracellular Cytokine Staining Assay Cells were plated at 5×10 ⁵ PBMCs per well in 96-well round-bottom plates and stimulated with HBV-derived peptide pools, 5 μg per peptide per mL final concentration. Plates were incubated for 20 hours at 37°C in a 5% _CO2 incubator. For the last 3 hours of the assay, PMA/Ionomycin was added to each well and Golgi plugs were added to all wells. Cells were harvested, washed with PBS+0.1% BSA (wash buffer) and stained with anti-CD3, anti-CD4 and anti-CD8 (BD Biosciences) for 30 minutes at 4°C. After further washing, cells were fixed and permeabilized with 100 μL of BD Cytofix/Cytoperm solution for 20 minutes at 4° C., followed by two washes with 1×BD Perm/Wash solution. Finally, cells were stained with anti-IL-2-FITC, anti-IFNγ-PE and anti-TNFα PerCP-Cy5.5 (BD Biosciences) for 30 minutes at 4°C. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences). Gating was based on medium-stimulated samples for each subject.

Determination of infecting HBV genotype
For HBV genotyping, a nested PCR method was first used followed by direct nucleotide sequencing. However, HBV genotype could not be determined using this method due to the low viral load in plasma from the majority of samples. The IMMUNIS® HBV genotype enzyme immunoassay (EIA) kit was then used. This assay used four genotype-dependent epitopes within the PreS2 region of HBsAg and was genotyped serologically by positive/negative combinations of four EIAs specific for each of the epitopes.

result
The first step in identifying regions of interest within the HBV proteome is the IFNγ ELISpot response of PBMCs from HBV-uninfected, unvaccinated healthy subjects and the sustained control phase of the disease. by comparing the IFNγ ELISpot responses of PBMCs from HBeAg-negative inactive carrier subjects with chronic HBV infection in ) and from HBeAg-negative subjects with chronic HBV infection on treatment there were. After short-term incubation with a library of overlapping short peptides (15-20 mers, 10-13 amino acid overlaps) representing approximately 70% of the HBV proteome, cytosolic sequences within the HBV polymerase, core, X and surface antigens, respectively, were obtained. PBMC were restimulated overnight with a pool of these short peptides representing specific regions of interest. IFNγ responses to these peptide pools were then assessed using a human IFNγ ELISpot assay.

Pools representing several antigenic regions were found to stimulate specific IFNγ responses in chronic HBV subjects. In particular, stimulation with pools representing terminal regions of HBV polymerase (pools 2 and 3) and pools representing regions of HBV core (pools 14-17) resulted in the greatest magnitude and population coverage in subjects with HBV infection. of IFNγ responses were produced (FIG. 1). To a lesser extent, pools 4-9 and pools 11-13 also tended to promote HBV-specific T cell responses.

To establish the role of infecting HBV genotype on the nature of HBV-specific responses to short peptide pools, infecting HBV genotype was determined for each subject. This was determined by means of HBV surface antigen epitope assessment in plasma samples. Both the IFNγ responses of PBMCs from subjects with HBV infection who are immunoregulated and from subjects with HBV infection who are undergoing treatment were then evaluated for HBV genotypes A, B, C and Grouped according to D. Some subjects were not classified into these genotypes due to limitations in assay sensitivity and the possible rarity of the sera evaluated. Therefore, these subjects were not included in this evaluation. The response profiles between the four genotypes showed similarities in that the regions showing the greatest magnitude of IFNγ response were generally in the terminal polymerase and core regions of the HBV proteome (Figure 2). Pools 2, 3, 10, 12, 14, 15, 16 and 17 appear to provide responses to multiple genotypes.

To establish the role of host subject genetic background on the nature of HBV-specific responses to short peptide pools, subjects in the study were grouped according to their ethnicity. The IFNγ responses of both PBMCs from immunoregulated HBV-infected subjects and from treated HBV-infected subjects were then analyzed across three broad ethnic groups, i.e., Comparisons were made among African/Arab, Caucasian and Asian/Indian. Response profiles between ethnic groups show similarities, again with the largest IFNγ responses with associated high population coverage found against pools derived from the terminal polymerase and core regions of the HBV proteome. (Fig. 3). Caucasians were found to have the highest average magnitude of responses to some pools compared to the other two ethnic groups in treated subjects compared to immunocontrolled subjects. Seemed to be slightly different in some respects. Pools 2, 3, 10, 14, 15, 16, 17 and 21 show a trend toward enhanced responses in multiple ethnic groups.

Finally, to further illustrate the type of IFNγ response by PBMCs to short peptide pools, short-term cultured cells were restimulated overnight for intracellular cytokine staining. Cells were then assessed by flow cytometry for CD3, CD4, CD8 and IFNγ expression. A comparison of IFNγ responses to short peptide pools 2 and 14 in PBMC from healthy and chronic HBV-infected subjects was performed (FIG. 4). These were two of the peptide pools that elicited the strongest HBV-specific responses in the IFNγ ELISpot assay. Consistent with the IFNγ ELISpot assay, we specifically found increased IFNγ expression in PBMC from subjects with chronic HBV infection. Furthermore, it was found to be a dual CD4 and CD8 T cell response.

[Example 2]
Evaluation of ex vivo immunogenicity of HBV-derived Densigen-related short peptide pools in human PBMCs.
Subjects with HBV Infection 104 subjects with clinically defined chronic HBV infection were enrolled in the Imperial Healthcare NHS Trust, Chelsea and Westminster Hospital NHS Foundation Trust, and Barts and the London NHS Trust in London. Enrolled in a protocol approved by the REC. After obtaining written informed consent from all subjects, fresh venous blood was collected and PBMCs and plasma were isolated and cryopreserved within 18 hours of blood collection. These subjects met the following criteria: good overall health, HBV-specific therapy: antiviral nucleoside (nucleotide) analog inhibitors and/or interferon therapy (clinically indicated). clinical status (chronic HBV infection, HBeAg-negative, and normal, persistent or intermittent elevation of ALT), HIV-negative, HCV-negative and HDV-negative.

healthy control subject
Cryopreserved PBMCs from 17 subjects were obtained from CTL Technologies. These subjects met the following criteria: good overall health, no vaccination against HBV, HBV surface antigen negative, HBV core antibody negative, HIV negative and HCV negative.

Short-Term Culture of PBMC One vial of PBMC from each subject (containing 1×10 ⁷ cells) was thawed and lymphocyte counts were determined using a Scepter™ handheld automated cell counter. PBMC were cultured in 24-well cell culture plates at a concentration of 1×10 ⁶ cells per mL in 2 mL of culture medium (CM: RPMI-1640 Glutamax supplemented with 5% human AB serum) for a total of 11 days. Cells were challenged with a peptide pool containing 144 overlapping HBV-derived short peptides (SEQ ID NOs:73-210 and SEQ ID NOs:142-147) ranging in length from 15-20 amino acids and overlapping by 10-13 amino acids. A final concentration of 0.1 μg per peptide per mL was used for stimulation. On day 4, IL-2 and IL-15 were added to the cultures to final concentrations of 10 IU/mL and 10 ng/mL, respectively. On day 10, cells were washed twice in CM and cultured for an additional day with 10 IU/mL IL-2. On day 11, cells were washed twice in CM, counted and incorporated into human IFNγ ELISpot assays or intracellular cytokine staining.

Human IFNγ ELISpot assay
96-well multiscreen PVDF filter plates (Millipore) were coated with 100 μl (1:80) of anti-human IFNγ capture mAb (R&D Systems) overnight at 4°C. Plates were then blocked with PBS supplemented with 1% BSA and 5% sucrose for 2 hours at 4°C. Cells were plated at 5×10 ⁴ PBMCs per well in triplicate wells. The final antigen concentrations used were: 23 short peptide pools related to Densigen from HBV (see below): 5 μg per peptide per mL, CEF peptide pool positive control: 1 μg per peptide per mL, PHA positive Control: 1 μg/mL. ELISpot plates were incubated for 18 hours at 37° C., 5% CO ₂ in a humidified environment. Plates were then washed and incubated with 100 μl (1:80) biotinylated anti-human IFNγ detection mAb (R&D Systems) for 2 hours at room temperature. After washing, plates were incubated with streptavidin-conjugated alkaline phosphatase (1:80) for 1 hour followed by substrate (30 minutes) according to the manufacturer's instructions (R&D Systems). Generated spots were counted using an automated plate counting system (CTL Europe).

Intracellular Cytokine Staining (ICS) Assay Cells were plated at 5×10 ⁵ PBMCs per well in 96-well round-bottom plates and stimulated with HBV-derived peptide pools at 5 μg per peptide per mL final concentration. Plates were incubated for 20 hours at 37°C in a 5% _CO2 incubator. For the last 3 hours of the assay, PMA/Ionomycin was added to each well and Golgi plugs were added to all wells. Cells were harvested, washed with PBS+0.1% BSA (wash buffer) and stained with anti-CD3, anti-CD4 and anti-CD8 (BD Biosciences) for 30 minutes at 4°C. After further washing, cells were fixed and permeabilized with 100 μL of BD Cytofix/Cytoperm solution for 20 minutes at 4° C., followed by two washes with 1×BD Perm/Wash solution. Finally, cells were stained with anti-IL-2-FITC, anti-IFNγ-PE and anti-TNFα PerCP-Cy5.5 (BD Biosciences) for 30 minutes at 4°C. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences). Gating was based on medium-stimulated samples for each subject.

Determination of infecting HBV genotype
For HBV genotyping, a nested PCR method was first used followed by direct nucleotide sequencing. However, HBV genotype could not be determined using this method due to the low viral load in plasma from the majority of samples. The IMMUNIS® HBV genotype enzyme immunoassay (EIA) kit was then used. This assay used four genotype-dependent epitopes within the PreS2 region of HBsAg and was genotyped serologically by positive/negative combinations of four EIAs specific for each of the epitopes.

result
A 35-40mer region of interest was identified after screening for responses to short peptide pools from HBV. These regions were further evaluated with the goal of using 35-40mer peptides in vaccines. Further evaluation involved redesigning previously used short peptide pools for restimulation after short-term culture. To more accurately reflect the peptides used in the vaccine, short-terminal peptides extending beyond the 35-40mer region of interest were removed from the pool. After short-term culture with the peptide library as before, these short peptide pools were then used for restimulation in human IFNγ ELISpot and ICS assays.

Restimulation with pools 24-46 showed predominant HBV-specific T cell responses against terminal polymerase-derived regions (pools 25 and 26) and core (pools 38 and 39 and pools 41-43) regions of the HBV proteome. (Fig. 5). HBV-specific responses were also found after stimulation with surface area pool 45. Regions of the polymerase corresponding to pool 28, pool 32, pool 33, pool 36 and pool 37 also generated significant T cell responses.

IFNγ ELISpot responses to pools 24-46 were grouped according to infecting HBV genotype (FIG. 6). Each of pools 27, 28, 29, 32, 35, 36 yields a predominant response to genotype C. Pools 25 and 26 yield predominant responses to genotype D. Pools 30 and 31 produce predominant responses to genotype B. Pools 38, 42, 43 and 44 yield predominant responses to genotype A. Some pools had more than one genotype: two genotypes for pools 26, 32, 33, 36 and 43, three genotypes for pools 37, 38, 41 and 42, or pools For 25, there is a tendency to promote responses to four genotypes.

IFNγ ELISpot responses to pools 24-46 were grouped according to infecting HBV genotype (FIG. 7). Pools 28, 29 and 30 yield predominant responses in Asian/Indian ethnicity. Pools 25, 33, 34, 35 and 37 produce predominant responses in Caucasians. Pools 38, 39, 41, 42 and 43 yield predominant responses in African/Arab ethnicity. Some pools had responses in more than one ethnic group: two ethnic groups for each of pools 26, 39 and 43, or three ethnic groups for each of pools 25, 38 and 42. tend to accelerate.

Results are summarized in Table 4 below.

Eight pools were selected for further analysis of T cell responses by intracellular cytokine staining. PBMC from 7-14 subjects (depending on the number of cells available after the IFNγ ELISpot assay) were stimulated overnight with 1 of 8 pools and the cells were enriched for surface CD3, Expression of CD4 and CD8 was co-stained for intracellular IFNγ, TNFα and IL-2 expression (FIG. 8). IFNγ expression was found in both CD8 and CD4 T cell populations, with responses ranging from 5/8 and 8/8 of the peptide pools evaluated, respectively. Similarly, TNFα expression was found in both CD8 and CD4 T cell populations, with a breadth of peptide pool responses of 3/8 and 6/8, respectively. It was found that CD8 T cells did not express IL-2 after stimulation with peptide pools, whereas CD4 T cells did express IL-2 after stimulation with 7 of the 8 pools.

[Example 3]
Construction of Fluorocarbon-Linked HBV Peptides Peptides having the amino acid sequences shown in SEQ ID NOS: 24, 25, 28, 33, 34, 36, 37 and 38 and 222 were subjected to FMOC (fluorenylmethyloxycarbonyl chloride) immobilized phase. Synthesized by synthesis. A fluorocarbon chain (C ₈ F ₁₇ (CH ₂ ) ₂ COOH) was then incorporated into the epsilon chain of an additional N-terminal lysine of each peptide to derive fluorocarbon-linked peptides. Cleavage in the presence of trifluoroacetic acid (TFA) and final purification by reverse-phase high-performance liquid chromatography (RP-HPLC) yielded purified fluorocarbon-linked or unmodified peptides. Purity of all preparations was greater than 90%.
FA-P113: K(FA)-VGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIK- _NH2 (SEQ ID NO: 24);
FA-P151: K(FA)-PEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASF- _NH2 (SEQ ID NO: 25);
FA-P376: K(FA)-KLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAISSVVRR- _NH2 (SEQ ID NO: 28);
FA-753(K): K(FA)-KKKEFGATVELLSFLPSDFFPSVRDLLDTASALYRKKK- _NH2 (SEQ ID NO:36);
FA-P856(K): K(FA)-LTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTKKK- _NH2 (SEQ ID NO:38);
FA-P877: K(FA)-PPAYRPPNAPILSTLPETTVVRRRGRSPRRR- _NH2 (SEQ ID NO:33);
FA-P277(K): K(FA)-RVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHKKK- _NH2 (SEQ ID NO:34),
FA-P797(K): K(FA)-SPHHTALRQAILSWGELMTLATWVGSNLEDPASRDKKK- _NH2 (SEQ ID NO:37);
FA-P1266(K): K(FA)-KKKGPLLVLQAGFFLLTRILTIPQSLDSW WTSLNFLKKK- _NH2 (SEQ ID NO: 222)
NP113: VGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIK (SEQ ID NO: 24);
NP151: PEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASF (SEQ ID NO: 25);
NP376: KLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAISSVVRR (SEQ ID NO:28);
NP753(K): KKKEFGATVELLSFLPSDFFPSVRDLLDTASALYRKKK (SEQ ID NO:36);
NP856(K): LTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTKKK (SEQ ID NO:38);
NP877: PPAYRPPNAPILSTLPETTVVRRRGRSPRRR (SEQ ID NO:33);
NP277(K): RVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHKKK (SEQ ID NO:34),
NP797(K): SPHHTALRQAILSWGELMTLATWVGSNLEDPASRDKKK (SEQ ID NO:37);
NP1266(K): KKKGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLKKK (SEQ ID NO: 222).

[Example 4]
Long HBV Peptide Formulations Vaccine candidate FP02.1, composed of nine fluorocarbon-conjugated HBV-derived peptides, prepared as described in Example 3, was formulated as follows. Conditions for peptide solubilization are described in Table 5. Briefly, each of the nine fluorocarbon-conjugated peptides was weighed into 5 ml glass vials. Each peptide was then solubilized using a 2-12% acetic acid solution in water to achieve a peptide concentration of 10 mg. After the peptide solutions (3.9 ml for each peptide) were blended in a 150 ml sterile container, 3.9 ml of 10% acetic acid solution in water was added. After 2 minutes of stirring using a magnetic stirrer, 39 mL of a 9.0% mannitol in water solution was added. After an additional 2 minutes of stirring using a magnetic stirrer, the solution was filtered using a 0.22 μm 33 mm Millex filter. 1.2 mL of the filtered solution was pumped into autoclaved 2 mL glass vials. Filtration recovery determined by RP-HPLC was >95%. Vials were frozen at -80°C for 1 hour. The samples were then freeze dried for 36 hours. Freeze-drying ventilation was performed under nitrogen and vials were capped at a pressure of 400-600 mbar. The amount of peptide was 600 μg peptide per vial and when reconstituted in 1.2 mL the final concentration was 500 μg peptide per ml.

[Example 5]
Preferred HBV peptides and mixtures are immunogenic in chronic HBV carriers regardless of disease stage, HBV viral genotype and subject ethnicity Methods and Materials Population Clinically identified as having chronic HBV infection Forty defined subjects were enrolled in REC-approved protocols at the Imperial Healthcare NHS Trust, Chelsea and Westminster Hospital NHS Foundation Trust, and Barts and the London NHS Trust in London. After obtaining written informed consent from all subjects, fresh venous blood was collected and PBMCs and plasma were isolated and cryopreserved within 18 hours of blood collection. These subjects met the following criteria: good overall health, HBV-specific therapy: antiviral nucleoside (nucleotide) analog inhibitors and/or interferon therapy (clinically indicated). clinical status (chronic HBV infection, HBeAg-negative, and normal, persistent or intermittent elevation of ALT), HIV-negative, HCV-negative and HDV-negative.

Short-Term Culture of PBMC One vial of PBMC from each subject (containing 1×10 ⁷ cells) was thawed and lymphocyte counts were determined using a Scepter™ handheld automated cell counter. PBMC were cultured in 24-well cell culture plates at a concentration of 1×10 ⁶ cells per mL in 2 mL of culture medium (CM: RPMI-1640 Glutamax supplemented with 5% human AB serum) for a total of 11 days. Cells were stimulated with a mixture of 9 HBV-derived long peptides described in Example 3.

Each peptide was used at a final concentration of 0.1 μg per peptide per mL. On day 4, IL-2 and IL-15 were added to the cultures to final concentrations of 10 IU/mL and 10 ng/mL, respectively. On day 10, cells were washed twice in CM and cultured for an additional day with 10 IU/mL IL-2. On day 11, cells were washed twice in CM, counted and incorporated into human IFNγ (interferon-gamma) ELISpot assays or intracellular cytokine staining.

Human IFNγ ELISpot assay
96-well multiscreen PVDF filter plates (Millipore) were coated with 100 μl (1:80) of anti-human IFNγ capture mAb (R&D Systems) overnight at 4°C. Plates were then blocked with PBS supplemented with 1% BSA and 5% sucrose for 2 hours at 4°C. Cells from short-term cultures were plated in triplicate wells at 5×10 ⁴ PBMCs per well. Final antigen concentrations used were 5 μg/mL for each peptide, PHA positive control: 1 μg/mL. ELISpot plates were incubated for 18 hours at 37° C., 5% CO ₂ in a humidified environment. Plates were then washed and incubated with 100 μl (1:80) biotinylated anti-human IFNγ detection mAb (R&D Systems) for 2 hours at room temperature. After washing, plates were incubated with streptavidin-conjugated alkaline phosphatase (1:80) for 1 hour followed by substrate (30 minutes) according to the manufacturer's instructions (R&D Systems). Generated spots were counted using an automated plate counting system (CTL Europe).

Intracellular Cytokine Staining Assay Cells from short-term cultures were plated at 5×10 ⁵ PBMCs per well in 96-well round-bottom plates and 9 HBV-derived long peptides (NP113, NP151, NP277(K), NP376 , NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)), final concentration 5 μg/mL. Plates were incubated for 20 hours at 37°C in a 5% _CO2 incubator. For the last 3 hours of the assay, PMA/Ionomycin was added to each well and Golgi plugs were added to all wells. Cells were harvested, washed with PBS+0.1% BSA (wash buffer) and stained with anti-CD3, anti-CD4 and anti-CD8 (BD Biosciences) for 30 minutes at 4°C. After further washing, cells were fixed and permeabilized with 100 μL of BD Cytofix/Cytoperm solution for 20 minutes at 4° C., followed by two washes with 1×BD Perm/Wash solution. Finally, cells were stained with anti-IL-2-FITC, anti-IFNγ-PE and anti-TNFα PerCP-Cy5.5 (BD Biosciences) for 30 minutes at 4°C. Samples were acquired on a FACSCanto II flow cytometer (BD Biosciences). Gating was based on medium-stimulated samples for each subject.

Determination of infecting HBV genotype
For HBV genotyping, a nested PCR method was first used followed by direct nucleotide sequencing. However, HBV genotype could not be determined using this method due to the low viral load in plasma from the majority of samples. The IMMUNIS® HBV genotype enzyme immunoassay (EIA) kit was then used. This assay used four genotype-dependent epitopes within the PreS2 region of HBsAg and was genotyped serologically by positive/negative combinations of four EIAs specific for each of the epitopes.

Results All peptides stimulated detectable T cell responses in both HBeAg-negative inactive carriers and HBV carriers in HBeAg-negative treated subjects (see Figures 9 and 10). Among the various peptides tested, NP113, NP151, NP376, NP753(K), NP797(K), NP856(K) and NP877 produced the highest levels of Response is facilitated. Surprisingly, the cumulative responses to NP113 and NP151 are higher in both populations than any other combination of the two peptides tested. Moreover, the cumulative response to NP113, NP151 and NP376 induces the highest level of response in both populations compared to any other combination of the three peptides tested. As shown in Figure 11, all of the tested peptides stimulate cross-reactive T cell responses across all four HBV genotypes. Peptides NP113, NP151, NP376, NP753(K), NP797(K), NP856(K) and NP877 across all four genotypes A, B, C and D, peptides NP2777(K) and NP1226(K) The highest response is promoted compared to Surprisingly, P113 stimulates the highest T cell responses across all four genotypes compared to all other peptides.

Figure 12 shows that all peptides stimulated T cell responses across all ethnic groups tested. Highest responses across all three ethnic groups with peptides NP113, NP151, NP376, NP753(K), NP797(K), NP856(K) and NP877 compared to NP277(K) and NP1266(K) is promoted.

Furthermore, all nine peptides demonstrate the ability to stimulate CD4 and/or CD8 T cell responses producing Th1 cytokines as measured by intracellular cytokine staining across all HBV genotypes (Figure 13).

[Example 6]
Advantages of Fluorocarbon-Conjugated Peptides Compared to Unconjugated Peptides in Their Ability to Promote T Cell Responses In Vivo Methods and Materials
The immunogenicity in mice of FP02.1 (containing 9 fluorocarbon-conjugated peptides) was compared to NP02.1 (containing 9 equivalent unconjugated peptides). Female BALB/c mice (n=7 per group) were treated with FP02.1 at a dose of 50 µg per peptide in a volume of 50 µL or with NP02.1 (unconjugated HBV peptides (NP113, NP151, NP277 (K ), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) at an equimolar dose of 43.8 μg per peptide in a volume of 50 μL (compared to FP02.1). Mice were immunized on day 0 and sacrificed on day 14. Splenocytes were immunized with a mixture of each of the 9 HBV peptides described in Example 3 at 5 μg per mL per peptide. was used in vitro for 18 hours in an ELISpot assay.

Alternatively, splenocytes were stimulated in vitro with 9 individual peptides at 5 μg per mL per peptide for 18 hours in an ELISpot assay. The number of IFNγ ⁺ spot-forming cells (SFC) was counted. Plates were then washed with PBS and incubated with a peroxidase-labeled antibody for IFNγ detection followed by incubation with substrate according to the manufacturer's instructions. Developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of IFNγ ⁺ SFC.

Results In mice immunized with a mixture of peptides conjugated to fluorocarbons (FP02.1), significantly more A large scale T cell response was observed (see Figure 14). Due to MHC restriction in the syngeneic BALC/C model, the immune response was dominated by four of the nine peptides contained in the vaccine (peptides NP113, NP151, NP376 and NP1266 (K ), see Figure 15).

Responses induced by FP02.1 were dominated by peptides NP113, NP151, NP376 and NP1266(K). Surprisingly, immune responses to peptides P113 and P376 were observed only with formulations containing fluorocarbon-conjugated peptides (see Figure 15).

In conclusion, conjugation of HBV-derived peptide sequences with fluorocarbon vectors promotes higher and broader T-cell responses compared to comparable unconjugated peptides.

[Example 7]
Fluorocarbon-Conjugated Peptides Promote CTL/CD8+ T Cell Responses Methods and Materials
The quality of the immune response induced by FP02.1 (containing nine fluorocarbon-conjugated peptides) was evaluated in mice. Female BALB/c mice (n=7 per group) were immunized intramuscularly with FP02.1 at a dose of 25 μg per peptide in a volume of 50 μL. Mice were immunized on day 0 and sacrificed on day 14.

Splenocytes were incubated in ELISpot assays with either CTL epitopes derived from peptide NP113 (CTL1 KYLPLDKGI) or CTL epitopes derived from NP151 (CTL2 HYFQTRHYL) at concentrations of 10 ¹ -10 ⁻⁹ μg/ml for 18 hours. stimulated in vitro. The number of IFNγ ⁺ SFC was counted. Plates were then washed with PBS and incubated with a peroxidase-labeled antibody for IFNγ detection followed by incubation with substrate according to the manufacturer's instructions. Developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of IFNγ ⁺ SFC.

Results As shown in Figure 16, FP02.1 promotes T cell responses against CTL epitopes restricted by MHC class I molecules after a single immunization.

[Example 8]
Synergy Between Fluorocarbon-Peptides Contained in the Same Formulation Methods and Materials Mice were administered alone or as part of a co-formulation with other fluorocarbon-conjugated peptides (FP02.1). The immunogenicity of FA-P113 was evaluated in mice. Female BALB/c mice (n=7 per group) were immunized intramuscularly with FA-P113 at a dose of 25 μg or with FP02.1 at a dose of 25 μg per peptide in a volume of 50 μL. Mice were immunized on day 0 and sacrificed on day 14. Splenocytes were stimulated in vitro with 5 μg/mL NP113 (not conjugated to fluorocarbon vector) in ELISpot assay for 18 hours. The number of IFNγ ⁺ SFC was counted. Plates were then washed with PBS and incubated with a peroxidase-labeled antibody for IFNγ detection followed by incubation with substrate according to the manufacturer's instructions. Developed spots were counted using an automated plate counting system (CTL Europe) to quantify the number of IFNγ ⁺ SFC.

Results A larger NP-113-specific T cell response was observed in mice immunized with a mixture of fluorocarbon-conjugated peptides (FP02.1) than in mice immunized with FA-P113 alone. (See Figure 17).

[Example 9]
Preferred HBV peptides and combinations contain epitopes with the ability to bind to a broad range of HLA class I molecules Methods and Materials
Using the ProImmune REVEAL binding assay, short peptides of 9 amino acids (HBV long peptides NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K) ) was determined for its ability to bind to one or more MHC class I alleles and stabilize MHC-peptide complexes. Detection is based on the presence or absence of the native conformation of the MHC-peptide complex. HLA class I alleles with high frequency (HLA-A*0201, A*0301, A*1101, A*2402, B*0702, B*0801, and B*3501) were selected. Binding to MHC molecules was compared to that of a known T-cell epitope, a positive control peptide with very strong binding properties. Each HBV peptide (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266 ( All potential nonamers for K)) were synthesized with >90% purity. Test peptide scores are reported quantitatively as a percentage of the signal generated by the positive control peptide, and peptides are indicated as having a putative pass/fail result. Good binders are considered peptides with a score of 45% against the positive control as defined by ProImmune.

Results The results, shown in Table 6, included each of the HBV long peptides (NP113, NP151, NP277, NP376, NP753, NP797, NP856, NP877 and The number of nonamers derived from NP1266) is indicated. All long HBV peptides contain at least 6 epitopes with the ability to bind at least 4 alleles. Any combination of the 6 long peptides contains nonamer epitopes with the ability to bind all alleles tested.

[Example 10]
Preferred HBV peptides and combinations contain epitopes with the ability to bind to a broad range of HLA class II molecules.
Using the ProImmune REVEAL® MHC-peptide binding assay, each of the HBV long peptides (NP113, NP151, NP277(K), NP376, NP753(K), NP797(K), NP856(K), NP877 and NP1266(K)) were determined for their ability to bind to one or more MHC class II alleles and stabilize MHC-peptide complexes. Frequent HLA class II alleles HLA-DR1 (α1*01:01; β1*01:01), HLA-DR15 (α1*01:01; β1*15:01), HLA-DR3 (α1*01: 01;β1*01:01), HLA-DR4(α1*01:01;β1*04:01), HLA-DR11(α1*01:01;β1*11:01), HLA-DR13(α1*01 :01; β1*13:01) and HLA-DR7 (α1*01:01; β1*07:01) were selected. Each peptide was given a score relative to a positive control peptide that is a known T cell epitope. Test peptide scores are reported quantitatively as a percentage of the signal generated by the positive control peptide, and peptides are indicated as having a putative pass/fail result. A good binder is considered to be a peptide with a Proimmune defined score of ≧15% of the positive control.

Results The results in Table 7 show that the HLA Binding scores across class II alleles are shown. Six out of nine HBV peptides bind to at least one HLA allele with a score of ≧15%. NP113, NP151 and NP376 bind four or more different HLA class II alleles. Surprisingly, P113 binds to all six alleles. The combination of peptides NP113 and NP877 binds to all tested HLA class II alleles.

The present disclosure includes the following embodiments.
[1] at least one of the sequences shown in SEQ ID NOs: 1 to 4 or a sequence that has at least 80% identity to one of the sequences shown in SEQ ID NOs: 1 to 4 A pharmaceutical composition comprising at least two peptides between 15 and 60 amino acids in length selected from peptides comprising a sequence of 15 contiguous amino acids, each peptide comprising at least one CD8+ T cell epitope and /or a pharmaceutical composition comprising at least one CD4+ T cell epitope, wherein each peptide induces a response in peripheral blood mononuclear cells (PBMC) from at least one individual with chronic HBV infection in an in vitro assay.
[2] The composition of embodiment 1, wherein said peptide is selected from peptides comprising at least 15 contiguous amino acids of one of the sequences set forth in SEQ ID NOS: 1-4.
[3] at least one peptide comprising at least 15 amino acids of one of the sequences shown in SEQ ID NOS: 1-3 and at least 15 amino acids of the sequence shown in SEQ ID NO: 4 3. A composition according to embodiment 1 or 2, comprising at least one peptide.
[4] at least one of the peptides is a sequence shown in one of SEQ ID NOS: 24-33, or at least 80 relative to one of the sequences shown in SEQ ID NOS: 24-33; The composition of any of embodiments 1-3, comprising sequences having % identity.
[5] At least one peptide further comprises one or more additional amino acids at the N-terminus and/or C-terminus to increase the net positive charge of the peptide and/or to decrease the hydrophobicity of the peptide. The composition of any of embodiments 1-4, comprising:
[6] The composition of embodiment 5, comprising a peptide comprising a sequence set forth in one of SEQ ID NOs:34-38.
[7] The composition of any one of embodiments 1-6, which is capable of inducing an immune response in PBMC from at least two individuals of different ethnicity and PBMC from two individuals with different HBV genotypes being infected. thing.
[8] among individuals infected with HBV genotype A, individuals infected with HBV genotype B, individuals infected with HBV genotype C, and individuals infected with HBV genotype D 8. The composition of embodiment 7, which is capable of eliciting an immune response in PBMC from 2, 3 or all individuals.
[9] The following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 67, 22 or 23;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 17, 18, 21 or 67;
(iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 16, 60, 19, 20 or 22;
(iv) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 20, 21, 67, 22 or 23;
9. The composition of embodiment 8, comprising at least one peptide selected from at least two of
[10] 2, 3, or HBV-infected Asian or Indian individuals, HBV-infected Caucasian individuals, and HBV-infected African or Arab individuals; A composition according to any of embodiments 7-9, which is capable of inducing an immune response in PBMC from all individuals.
[11] The following groups:
(i) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 16, 60, 17, 18, 20, 21, 67 or 22;
(ii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 19, 20, 22 or 23;
(iii) a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 14, 15, 20, 21, 67, 22 or 23;
11. The composition of embodiment 10, comprising at least one peptide selected from at least two of
[12] Any of embodiments 1-11, comprising one or more peptides of 15-60 amino acids in length comprising a fragment of at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO:1. The described composition.
[13] The composition of embodiment 12, comprising a peptide comprising at least 15 contiguous amino acids of the sequence shown in SEQ ID NO:5, 6, 14 or 15.
[14] The composition of embodiment 12 or 13, comprising a peptide comprising a sequence set forth in one of SEQ ID NOS: 80, 81, 82, 83, 86, 87, 88 or 89.
[15] The composition of any of embodiments 12-14, comprising a peptide comprising the sequence shown in SEQ ID NO:24 or 25.
[16] The composition of any of embodiments 1-15, comprising from 2 to 10 of said peptides.
[17] Embodiments 1-16 comprising at least two of the ten peptides comprising the sequences set forth in SEQ ID NOs: 24, 25, 27, 28, 33, 34, 35, 36, 37 and 38 The composition according to any one of
[18] a sequence of at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO: 55, or at least 80% identity to at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO: 55; at least one peptide of 15 to 60 amino acids in length comprising a sequence having , further comprising at least one peptide that elicits a response in peripheral blood mononuclear cells (PBMCs) from at least one individual with chronic HBV infection. .
[19] The composition of embodiment 18, wherein the peptide comprises the sequence shown in SEQ ID NO:221 or SEQ ID NO:222.
[20] The composition of embodiment 19, comprising a peptide comprising the sequences set forth in SEQ ID NOS: 24, 25, 28, 33, 34, 36, 37, 38 and 222.
[21] The composition of any of embodiments 1-20, wherein said peptide is linked to a fluorocarbon vector.
[22] The composition of any of embodiments 1-21, further comprising an HBc antigen, HBe antigen, or HBs antigen.
[23] The composition of any of embodiments 1-22, further comprising an adjuvant.
[24] A composition according to any of embodiments 1-23 for use in treating or preventing HBV infection.
[25] The composition of embodiment 21, for use in treating HBeAg-negative patients.
[26] The composition of embodiment 21, for use in treating HBeAg-positive patients.
[27] (i) interferon-alpha and/or nucleoside/nucleotide analogues (NUC), and/or (ii) anti-PD1 blocking antibodies, anti-PD1L blocking antibodies, anti-LAG3 blocking antibodies, anti-TIM3 blocking antibodies, anti-CTLA4 blocking antibodies A composition according to any of embodiments 21-23 for use according to any of embodiments 20-22 in combination with an antibody and/or cyclophosphamide.
[28] The composition of any of embodiments 21-24, wherein said treatment results in HBsAg elimination or HBsAg seroconversion.
[29] A composition according to any of embodiments 1-20 for use in the treatment or prevention of end-stage liver disease or hepatocellular carcinoma.
[30] A composition according to any of embodiments 1-20 for use in treating or preventing hepatitis D virus (HDV) infection.
[31] at least a sequence set forth in any one of SEQ ID NOs: 1-4 or a sequence having at least 80% identity to one of the sequences set forth in SEQ ID NOs: 1-4 A peptide 15-60 amino acids in length comprising 15 contiguous amino acids and comprising at least one CD8+ T-cell epitope and/or at least one CD4+ T-cell epitope, wherein in an in vitro assay, chronic HBV infection A peptide that is capable of inducing a response in peripheral blood mononuclear cells (PBMC) from at least one individual with disease.
[32] The peptide of embodiment 28, comprising at least 15 contiguous amino acids of the sequence set forth in SEQ ID NO:5, 6, 14 or 15.
[33] comprising a sequence having at least 80% identity to one of the sequences set forth in SEQ ID NOs:24-38 or to one of the sequences set forth in SEQ ID NOs:24-38 peptide.
[34] The peptide of any of embodiments 28-30, which is covalently linked to a fluorocarbon vector.
[35] A method of treating or preventing an HBV infection, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any of embodiments 1-20.
[36] Use of the composition of any of embodiments 1-20 in the manufacture of a medicament for treating or preventing HBV.

Claims (13)

A pharmaceutical composition comprising a combination of immunogenic peptides of 25-60 amino acids in length having at least 15 contiguous amino acids of the HBV genotype A, B, C, or D consensus sequence from HBV polymerase, core protein or surface antigen. wherein each peptide is covalently linked to a carbon vector for intracellular delivery of the peptide, the carbon vector comprising a chain comprising 3-30 carbon atoms, at least one of which is substituted by fluorine, chlorine, bromine or iodine, the composition comprising:
As a combination of said immunogenic peptides,
a first peptide comprising the amino acid sequence shown in SEQ ID NO: 24;
a second peptide comprising the amino acid sequence shown in SEQ ID NO: 25;
a third peptide comprising the amino acid sequence shown in SEQ ID NO: 28;
a fourth peptide comprising the amino acid sequence shown in SEQ ID NO: 33;
a fifth peptide comprising the amino acid sequence shown in SEQ ID NO: 34;
a sixth peptide comprising the amino acid sequence shown in SEQ ID NO: 36;
a seventh peptide comprising the amino acid sequence shown in SEQ ID NO: 37;
an eighth peptide comprising the amino acid sequence shown in SEQ ID NO:38 and a ninth peptide comprising the amino acid sequence shown in SEQ ID NO:222;
The pharmaceutical composition comprising: 2. The composition of claim 1, further comprising an adjuvant. The peptide linked to the carbon vector has _the chemical structure _{CmFn - CyHx-} (Sp)-R
[Wherein m = 3 to 30, n ≤ 2m + 1, y = 0 to 15, x ≤ 2y, (m + y) = 3 to 30, Sp is optional and R is said immunogenic peptide]
The composition of claim 1, comprising: 2. The composition of claim 1, wherein said peptide combination is present in solution at a concentration of about 0.1 mM to about 10 mM. 2. The composition of claim 1, wherein the composition is in dry form. A composition according to any one of claims 1 to 5 for treating or preventing HBV infection. A pharmaceutical composition comprising a combination of immunogenic peptides 25-60 amino acids in length having at least 15 contiguous amino acids of the HBV genotype A, B, C, or D consensus sequence from HBV polymerase, core protein or surface antigen and
As a combination of said immunogenic peptides,
a first peptide comprising the amino acid sequence shown in SEQ ID NO: 24;
a second peptide comprising the amino acid sequence shown in SEQ ID NO: 25;
a third peptide comprising the amino acid sequence shown in SEQ ID NO: 28;
a fourth peptide comprising the amino acid sequence shown in SEQ ID NO: 33;
a fifth peptide comprising the amino acid sequence shown in SEQ ID NO: 34;
a sixth peptide comprising the amino acid sequence shown in SEQ ID NO: 36;
a seventh peptide comprising the amino acid sequence shown in SEQ ID NO: 37;
an eighth peptide comprising the amino acid sequence shown in SEQ ID NO:38 and a ninth peptide comprising the amino acid sequence shown in SEQ ID NO:222;
including
Each _peptide has _the chemical structure _CmFn - _CyHx- (Sp)-R
[Wherein m = 3 to 30, n ≤ 2m + 1, y = 0 to 15, x ≤ 2y, (m + y) = 3 to 30, Sp is optional and R is said immunogenic peptide],
The pharmaceutical composition linked to a fluorocarbon chain having fluorocarbon chains

8. The composition of claim 7, selected from _8. The composition of claim 7, wherein the fluorocarbon chain has _C8F17 ( _CH2 ) ₂ (Sp)-R. 8. The composition of Claim 7, further comprising an adjuvant. 8. The composition of claim 7, wherein said peptide combination is present in solution at a concentration of about 0.1 mM to about 10 mM. 8. A composition according to claim 7, wherein the composition is in dry form. A composition according to any one of claims 7 to 12 for treating or preventing HBV infection.

Images